WO2001044452A1 - Subtilase variants having an improved wash performance on egg stains - Google Patents

Abstract

The present invention relates to the use of a subtilase variant for removal of egg stains from laundry or from hard surfaces, where the subtilase variant comprises at least one additional amino acid residue in the active site loop (b) region from position 95 to 103 (BASBPN numbering). These subtilase variants are useful exhibiting excellent or improved wash performance on egg stains when used in e.g. cleaning or detergent compositions, including automatic dishwash compositions. The present invention also relates to novel subtilase variants, to isolated DNA sequences encoding the variants, expression vectors, host cells, and methods for producing and using the variants of the invention. Further, the present invention relates to cleaning and detergent compositions comprising the variants of the invention.

Claims

1SUBTILASE VARIANTS HAVING AN IMPROVED WASH PERFORMANCE ON EGG STAINSTECHNICAL FIELD The present invention relates to the use of subtilase variants for removal of egg stains from laundry or from hard surfaces. In particular the present invention relates to the use of a subtilase variant for removal of egg stains from laundry or from hard surfaces, where the subtilase variant comprises at least one additional amino acid residue in the active site loop (b) region from position 95 to 103 (BASBPN numbering, vide infra) . These subtilase variants are useful exhibiting excellent or improved wash performance on egg stains when used in e.g. cleaning or detergent compositions, such as laundry detergent compositions and dishwash composition, including automatic dishwash compositions. The present invention also relates to novel subtilase variants, to isolated DNA sequences encoding the variants, expression vectors, host cells, and methods for producing and using the variants of the invention. Further, the present invention relates to cleaning and detergent compositions comprising the variants of the invention.BACKGROUND OF THE INVENTIONIn the detergent industry enzymes have for more than 30 years been implemented in washing formulations. Enzymes used in such formulations comprise proteases, lipases, amylases, cellulases, as well as other enzymes, or mixtures thereof. Commercially most important enzymes are proteases.An increasing number of commercially used proteases are protein engineered variants of naturally occurring wild type proteases, e.g. DURAZYM® (Novo Nordisk A/S) , RELASE® (Novo Nordisk A/S) , 2 MAXAPEM® (Gist-Brocades N.V.), PURAFECT® (Genencor International, Inc . ) .Further, a number of protease variants are described in the art. A thorough list of prior art protease variants is given in WO 99/27082.However, even though a number of useful protease variants have been described, there is still a need for new improved proteases or protease variants for a number of industrial uses.In particular, the problem of removing egg stains from e.g. laundry or hard surfaces has been pronounced due to the fact that many proteases are inhibited by substances present in the egg white. Examples of such substances include trypsin inhibitor type IV-0 (Ovo-inhibitor) and trypsin inhibitor type III-O (Ovomucoid) .Therefore, an object of the present invention, is to provide improved subtilase variants, which are not, or which are only to a limited extent, inhibited by such substances. A further object of the present invention is to provide improved subtilase variants, which are suitable for removal of egg stains from, for example, laundry and/or hard surfaces.SUMMARY OF THE INVENTIONThus, in a first aspect the present invention relates to the use of a subtilase variant for removal of egg stains from laundry or from hard surfaces, the subtilase variant comprising at least one additional amino acid residue in the active site loop (b) region from position 95 to 103 (BASBPN numbering) . 3In a second aspect the present invention relates to a subtilase variant selected from the group consisting ofa variant comprising at least one additional amino acid residue in the ac- tive site (b) loop corresponding to the insertion of at least one additional amino acid residue between positions 98 and 99 and further coπprising at least one additional modification (BASBPN numbering) , anda variant comprising at least one additional amino acid residue in the ac- tive site (b) loop corresponding to the insertion of at least one additional amino acid residue between positions 99 and 100 and further comprising at least one additional modification (BASBPN numbering) ,where the variant - when tested in the "Ovo-inhibition Assay" dis- closed in Example 4 herein - has a Residual Activity of at least 10%.In a third aspect the present invention relates to a subtilase variant selected from the group consisting ofa variant comprising an insertion of at least one additional amino acid residue between positions 98 and 99 and further comprising a substitution in positions 133 and 143,a variant comprising an insertion of at least one additional amino acid residue between positions 99 and 100 and further comprising a substitution in position 99,a variant comprising an insertion of at least one additional amino acid residue between positions 98 and 99 and further coπprising substitutions in positions 167, 170 and 194, 4 a variant coπprising an insertion of at least one additional aπiino acid residue between positions 99 and 100 and further coπprising an insertion of at least one additional amino acid residue between positions 216 and 217,a variant coπprising an insertion of at least one additional amino acid residue between positions 99 and 100 and further coπprising an insertion of at least one additional amino acid residue between positions 217 and 218,a variant coπprising an insertion of at least one additional amino acid residue between positions 99 and 100 and further coπprising an insertion of at least one additional amino acid residue between positions 42 and 43, anda variant coπprising an insertion of at least one additional aπ no acid residue between positions 99 and 100 and further coπprising an insertion of at least one additional arπino acid residue between positions 129 and 130.In a fourth aspect the present invention relates to an isolated DNA sequence encoding a subtilase variant of the invention.In a fifth aspect the present invention relates to an expression vector coπprising the isolated DNA sequence of the invention.In a sixth aspect the present invention relates to a microbial host cell transformed with the expression vector of the invention.In a seventh aspect the present invention relates to a method for producing a subtilase variant according to the invention, wherein a host accord- 5 ing to the invention is cultured under conditions conducive to the expression and secretion of said variant, and the variant is recovered.In an eight aspect the present invention relates to a cleaning or detergent composition, preferably a laundry or dishwash composition, comprising the variant of the invention.In a ninth aspect the present invention relates to a method for removal of egg stains from a hard surface or from laundry, the method comprising contacting the egg stain-containing hard surface or the egg stain-containing laundry with a cleaning or detergent composition, preferably a laundry or dishwash composition, containing a subtilase variant comprising at least one additional amino acid residue in the active site loop (b) region from position 95 to 103 (BASBPN numbering) .Still other aspect of the present invention will be apparent from the below description and from the appended claims.Concerning alignment and numbering reference is made to Fig. 1 which shows an alignments between subtilisin BPN' (a) (BASBPN) and subtilisin 309 (BLSAVI) (b) .These alignments are in this patent application used as a reference for numbering the residues.DEFINITONSPrior to discussing this invention in further detail, the following terms and conventions will first be defined.NOMENCLATURE AND CONVENTIONS FOR DESIGNATION OF VARIANTS 6In describing the various subtilase enzyme variants produced or contemplated according to the invention, the following nomenclatures and conventions have been adapted for ease of reference :A frame of reference is first defined by aligning the isolated or parent enzyme with subtilisin BPN' (BASBPN) .The alignment can be obtained by the GAP routine of the GCG package version 9.1 to number the variants using the following parameters: gap creation penalty = 8 and gap extension penalty = 8 and all other parameters kept at their default values.Another method is to use known recognized alignments between subtilases, such as the alignment indicated in WO 91/00345. In most cases the differences will not be of any importance.Thereby a number of deletions and insertions will be defined in relation to BASBPN. In Fig. 1, subtilisin 309 (Savinase®) has 6 deletions in positions 36, 58, 158, 162, 163, and 164 in comparison to BASBPN. These deletions are in Fig. 1 indicated by asterixes (*) .The various modifications performed in a parent enzyme is indicated in general using three elements as follows:Original amino acid position substituted amino acidThe notation G195E thus means a substitution of a glycine in position 195 with a glutamic acid. 7In the case where the original amino acid residue may be any amino acid residue, a short hand notation may at times be used indicating only the position and substituted amino acid:Position substituted amino acidSuch a notation is particular relevant in connection with modification (s) in homologous subtilases ( vide infra) .Similarly when the identity of the substituting amino acid residue (s) is immaterial:Original amino acid positionWhen both the original amino acid(s) and substituted amino acid(s) may comprise any amino acid, then only the position is indicated, e.g.: 170.When the original amino acid(s) and/or substituted amino acid(s) may comprise more than one, but not all amino acid(s), then the selected amino acids are indicated inside brackets :Original amino acid position {substituted amino acid , . . . , substituted amino acidn)For specific variants the specific three or one letter codes are used, including the codes Xaa and X to indicate any amino acid residue .SUBSTITUTIONS: The substitution of glutamic acid for glycine in position 195 is designated as : 8 Glyl95Glu or G195Eor the substitution of any amino acid residue acid for glycine in position 195 is designated as:Glyl95Xaa or G195X orGlyl95 or G195The substitution of serine for any amino acid residue in position 170 would thus be designatedXaal70Ser or X170S. or 170Ser or 170SSuch a notation is particular relevant in connection with modification (s) in homologous subtilases ( vide infra) . 170Ser is thus meant to comprise e . g. both a Lysl70Ser modification in BASBPN and Argl70Ser modification in BLSAVI (cf . Fig. 1) .For a modification where the original amino acid(s) and/or substituted amino acid(s) may comprise more than one, but not all amino acid(s), the substitution of glycine, alanine, serine or threonine for arginine in position 170 would be indicated byArgl70{Gly,Ala,Ser,Thr} or R170 {G, A, S, T}to indicate the variantsR170G, R170A, R170S, and R170T. 9DELETIONS :A deletion of glycine in position 195 will be indicated by:Glyl95* or G195*Correspondingly the deletion of more than one amino acid residue, such as the deletion of glycine and leucine in positions 195 and 196 will be designatedGlyl95*+Leul96* or G195AL196*INSERTIONS :The insertion of an additional amino acid residue such as e.g. a lysine after G195 is indicated by:Glyl95GlyLys or G195GK; or, when more than one amino acid residue is inserted, such as e.g. a Lys, Ala and Ser after G195 this will be indicated as:. Glyl95GlyLysAlaSer or G195GKAS (SEQ ID NO:l)In such cases the inserted amino acid residue (s) are numbered by the addition of lower case letters to the position number of the amino acid residue preceding the inserted amino acid residue (s) . In the above example the sequences 194 to 196 would thus be:194 195 196 BLSAVI A - G - L194 195 195a 195b 195c 196 Variant A - G - K - A - S - L (SEQ ID NO: 16) 10In cases where an amino acid residue identical to the existing amino acid residue is inserted it is clear that a degeneracy in the nomenclature arises. If for example a glycine is inserted after the glycine in the above example this would be indicated by G195GG. The same actual change could just as well be indicated as A194AG for the change from194 195 196 BLSAVI A - G - Lto194 195 195a 196 Variant A - G - G - L (SEQ ID NO: 27) 194 194a 195 196Such instances will be apparent to the skilled person, and the indication G195GG and corresponding indications for this type of insertions are thus meant to comprise such equivalent degenerate indications.FILLING A GAP:Where a deletion in an enzyme exists in the reference comparison with the subtilisin BPN' sequence used for the numbering, an insertion in such a position is indicated as:*36Asp or *36Dfor the insertion of an aspartic acid in position 36MULTIPLE MODIFICATIONS 11Variants comprising multiple modifications are separated by pluses , e.g. :Argl70Tyr+Glyl95Glu or R170Y+G195Erepresenting modifications in positions 170 and 195 substituting tyrosine and glutamic acid for arginine and glycine, respectively.Thus, Tyrl67{Gly,Ala,Ser,Thr}+Argl70{Gly,Ala, Ser,Thr} designates the following variants:Tyrl67Gly+Argl70Gly, Tyrl67Gly+Argl70Ala, Tyrl67Gly+Argl70Ser, Tyr167Gly+Argl7OThr, Tyrl67Ala+Argl70Gly, Tyrl67Ala+Argl70Ala, Tyrl67Ala+Argl70Ser, Tyr167A1a+Argl7OThr, Tyrl67Ser+Argl70Gly, Tyrl67Ser+Argl70Ala, Tyrl67Ser+Argl70Ser, Tyrl67Ser4-Argl70Thr, Tyrl67Thr+Argl70Gly, Tyrl67Thr+Argl70Ala, Tyrl67Thr+Argl70Ser, and Tyr167Thr+Argl7OThr .This nomenclature is particular relevant relating to modifications aimed at substituting, replacing, inserting or deleting amino acid residues having specific common properties, such as residues of positive charge (K, R, H) , negative charge (D, E) , or conservative amino acid modification (s) of e.g. Tyrl67{Gly,Ala,Ser,Thr}+Argl70{Gly,Ala,Ser,Thr} , which signifies substituting a small amino acid for another small amino acid. See section "Detailed description of the invention" for further details.Proteases 12Enzymes cleaving the amide linkages in protein substrates are classified as proteases, or (interchangeably) peptidases (see Walsh, 1979, Enzymatic Reaction Mechanisms . W.H. Freeman and Company, San Francisco, Chapter 3) .Numbering of amino acid positions/residuesIf nothing else is mentioned the amino acid numbering used herein correspond to that of the subtilase BPN' (BASBPN) sequence. For further description of the BPN' sequence, see Fig. 1 or Siezen et al . , Protein Engng. 4 (1991) 719-737.Serine proteasesA serine protease is an enzyme which catalyzes the hydrolysis of peptide bonds, and in which there is an essential serine residue at the active site (White, Handler and Smith, 1973 " Principles of Biochemistry , " Fifth Edition, McGraw-Hill Book Company, NY, pp. 271-272) .The bacterial serine proteases have molecular weights in the 20,000 to 45,000 Dalton range. They are inhibited by diisopro- pylfluorophosphate . They hydrolyze simple terminal esters and are similar in activity to eukaryotic chymotrypsin, also a serine protease. A more narrow term, alkaline protease, covering a sub-group, reflects the high pH optimum of some of the serine proteases, from pH 9.0 to 11.0 (for review, see Priest (1977) Bacteriological Rev. 41 711-753) .SubtilasesA sub-group of the serine proteases tentatively designated subtilases has been proposed by Siezen et al . , Protein Engng. 4 (1991) 719-737 and Siezen et al . Protein Science 6 (1997) 501- 523. They are defined by homology analysis of more than 170 13 amino acid sequences of serine proteases previously referred to as subtilisin-like proteases. A subtilisin was previously often defined as a serine protease produced by Gram-positive bacteria or fungi, and according to Siezen et al . now is a subgroup of the subtilases. A wide variety of subtilases have been identified, and the amino acid sequence of a number of subtilases has been determined. For a more detailed description of such subtilases and their amino acid sequences reference is made to Siezen et al . (1997).One subgroup of the subtilases, I -SI or "true" subtilisins, comprises the "classical" subtilisins, such as subtilisin 168 (BSS168) , subtilisin BPN' (SEQ ID NO:38), subtilisin Carlsberg (ALCALASE®, NOVO NORDISK A/S) , and subtilisin DY (BSSDY) .A further subgroup of the subtilases, I-S2 or high alkaline subtilisins, is recognized by Siezen et al . ( supra) . Sub-group I-S2 proteases are described as highly alkaline subtilisins and comprises enzymes such as subtilisin PB92 (BAALKP) (MAXACAL*, Gist-Brocades NV) , subtilisin 309 (SEQ ID NO-.49) (SAVINASE®, NOVO NORDISK A/S) , subtilisin 147 (BLS147) (ESPERASE , NOVO NORDISK A/S) , and alkaline elastase YaB (BSEYAB) ."SAVINASE®" SAVINASE® is marketed by NOVO NORDISK A/S. It is subtilisin 309 from B . Lentus and differs from BAALKP only in one position (N87S, see Fig. 1 herein) . SAVINASE® has the amino acid sequence designated b) in Fig. 1 and as shown in SEQ ID NO: 49.Parent subtilaseThe term "parent subtilase" describes a subtilase defined according to Siezen et al . (1991 and 1997) . For further details 14 see description of "SUBTILASES" immediately above. A parent subtilase may also be a subtilase isolated from a natural source, wherein subsequent modifications have been made while retaining the characteristic of a subtilase. Furthermore, a parent subtilase may also be a subtilase which has been prepared by the DNA shuffling technique, such as described by J.E. Ness et al . , Nature Biotechnology, 17, 893-896 (1999). Alternatively the term "parent subtilase" may be termed "wild type subtilase" .Modification (s) of a subtilase variantThe term "modification (s) " used herein is defined to include chemical modification of a subtilase as well as genetic manipulation of the DNA encoding a subtilase. The modification (s) can be replacement (s) of the amino acid side chain (s), substitution (s) , deletion (s) and/or insertions in or at the amino acid(s) of interest.Subtilase variant In the context of this invention, the term subtilase variant or mutated subtilase means a subtilase that has been produced by an organism which is expressing a mutant gene derived from a parent microorganism which possessed an original or parent gene and which produced a corresponding parent enzyme, the parent gene having been mutated in order to produce the mutant gene from which said mutated subtilase protease is produced when expressed in a suitable host . 15Homologous subtilase sequencesSpecific active site loop regions, and amino acid insertions in said loops of SAVINASE® subtilase are identified for modification herein to obtain a subtilase variant of the invention.However, the invention is not limited to modifications of this particular subtilase, but extend to other parent (wild-type) subtilases, which have a homologous primary structure to that of SAVINASE®. The homology between two amino acid sequences is in this context described by the parameter "identity" .In order to determine the degree of identity between two subtilases the GAP routine of the GCG package version 9.1 can be ap- plied ( infra) using the same settings. The output from the routine is besides the amino acid alignment the calculation of the "Percent Identity" between the two sequences.Based on this description it is routine for a person skilled in the art to identify suitable homologous subtilases and corresponding homologous active site loop regions, which can be modified according to the invention.Isolated DNA sequence The term "isolated", when applied to a DNA sequence molecule, denotes that the DNA sequence has been removed from its natural genetic milieu and is thus free of other extraneous or unwanted coding sequences, and is in a form suitable for use within genetically engineered protein production systems. Such isolated molecules are those that are separated from their natural environment and include cDNA and genomic clones. Isolated DNA molecules of the present invention are free of other genes with 16 which they are ordinarily associated, but may include naturally occurring 5 ' and 3 ' untranslated regions such as promoters and terminators. The identification of associated regions will be evident to one of ordinary skill in the art (see for example, Dynan and Tijan, Nature 31_6: 774-78, 1985) . The term "an isolated DNA sequence" may alternatively be termed "a cloned DNA sequence" .Isolated protein When applied to a protein, the term "isolated" indicates that the protein has been removed from its native environment.In a preferred form, the isolated protein is substantially free of other proteins, particularly other homologous proteins (i.e. "homologous impurities" (see below) ) .An isolated protein is more than 10% pure, preferably more than 20% pure, more preferably more than 30% pure, as determined by SDS-PAGE. Further it is preferred to provide the protein in a highly purified form, i.e., more than 40% pure, more than 60% pure, more than 80% pure, more preferably more than 95% pure, and most preferably more than 99% pure, as determined by SDS- PAGE.The term "isolated protein" may alternatively be termed "purified protein" .Homologous impuritiesThe term "homologous impurities" means any impurity (e.g. another polypeptide than the subtilase of the invention) , which originate from the homologous cell where the subtilase of the invention is originally obtained from. 17Obtained fromThe term "obtained from" as used herein in connection with a specific microbial source, means that the polynucleotide and/or 5 subtilase produced by the specific source, or by a cell in which a gene from the source has been inserted.SubstrateThe term "substrate" used in connection with a substrate for a io protease should be interpreted in its broadest form as comprising a compound containing at least one peptide bond susceptible to hydrolysis by a subtilisin protease.Product 15 The term "product" used in connection with a product derived from a protease enzymatic reaction should, in the context of the present invention, be interpreted to include the products of a hydrolysis reaction involving a subtilase protease. A product may be the substrate in a subsequent hydrolysis reaction.20Wash PerformanceIn the present context the term "wash performance" is used as an enzyme's ability to remove egg stains present on the object to be cleaned during e.g. wash or hard surface cleaning. See also 25 the "Model Detergent Wash Performance Test" in Example 3 herein.Performance FactorThe term "Performance Factor" is defined with respect to the below formula-> '-) " — -Aariant — •'"•■parent 18 wherein P is the Performance Factor, Rvarιarιt is the reflectance (measured at 460 nm) of the test material after being treated with a subtilase variant as described in the "Model Detergent Wash Performance Test", and Rparent is the reflectance (measured at 460 nm) of the test material after being treated with the corresponding parent subtilase as described in the "Model Detergent Wash Performance Test". For further details, see the "Model Detergent Wash Performance Test" in Example 3 herein.Residual ActivityThe term "Residual Activity" is defined as described in the "Ovo-inhibition Assay" herein (see Example 4) .BRIEF DESCRIPTION OF THE DRAWING Fig. 1 shows an alignment between subtilisin BPN' (a) and®Savinase (b) using the GAP routine mentioned above.DETAILED DESCRIPTION OF THE INVENTIONThe present inventors have found that subtilisin variants, wherein the active site loop (b) region is longer than those presently known, exhibit improved wash performance with respect to removal of egg stains. The identification thereof was done in constructing subtilisin variants, especially of the subtilisin 309 (BLSAVI or Savinase®) , exhibiting improved wash performance properties (with respect to removal of egg stains) in model detergent compositions relative to the parent wild type enzyme.Without being limited to any specific theory it is presently believed that the improved effect is due to an impeded binding of the egg white inhibitor in the active site loop (b) region of the subtilase variant. This in turn is probably due to structural changes of the active site loop (b) region because of 19 insertion of one or more additional amino acid residues in this particular site of the enzyme.Thus, variants which are contemplated as being suitable for the uses described herein are such variants where, when compared to the wild- type subtilase, one or more amino acid residues has been inserted in one or more of the following positions: between positions 95 and 96, between positions 96 and 97, between positions 97 and 98, between positions 98 and 99, between positions 99 and 100, between positions 100 and 101, between positions 101 and 102, between positions 102 and 103, between positions 103 and 104, and combinations thereof .Preferably, the insertion is made between position 97 and 98, between po- sitions 98 and 99, between positions 99 and 100 and/or between positions 100 and 101, in particular between positions 98 and 99 and between positions 99 and 100.A subtilase variant of the first aspect of the invention may be a parent or wild-type subtilase identified and isolated from nature .Such a parent wildtype subtilase may be specifically screened for by standard techniques known in the art .One preferred way of doing this may be by specifically PCR amplify DNA regions known to encode active site loops in subtilases from numerous different microorganism, preferably different Bacillus strains. 20Subtilases are a group of conserved enzymes, in the sense that their DNA and amino acid sequences are homologous . Accordingly it is possible to construct relatively specific primers flanking active site loops.One way of doing this is by investigating an alignment of different subtilases (see e.g. Siezen et al . Protein Science 6 (1997) 501-523) . It is from this routine work for a person skilled in the art to construct PCR primers flanking the active site loop corresponding to the active site loop (b) between amino acid residue 95 to 103 in any of the group I-Sl or I-S2 groups, such as from BLSAVI. Using such PCR primers to amplify DNA from a number of different microorganism, preferably different Bacillus strains, followed by DNA sequencing of said amplified PCR fragments, it will be possible to identify strains which produce subtilases of these groups comprising a longer, as compared to e.g. BLSAVI, active site region corresponding to the active site loop region from positions 95 to 103. Having identified the strain and a partial DNA sequence of such a subtilase of interest, it is routine work for a person skilled in the art to complete cloning, expression and purification of such a subtilase.However, it is envisaged that a subtilase variant of the invention predominantly is a variant of a parent subtilase.A subtilase variant suitable for the uses described herein, may be constructed by standard techniques known in the art such as by site-directed/random mutagenesis or by DNA shuffling of different subtilase sequences. See the "Material and Methods" section herein ( vide infra) for further details. 21As will be acknowledged by the skilled person, the variants described herein may, in addition to the at least one insertion from position 95 to 103, comprise at least one further modification. For example, the variants may comprise one or more substitutions in the active site loop (b) region as well as one or more substitutions outside said region. Furthermore, the variants may comprise one or more further insertions outside the active site loop (b) region.Moreover, the insertions in the regions described herein may encompass insertion of more than just one amino acid residue. For example the variant according to the invention may contain one insertion, two insertions, or more than two insertions, such as three, four or five insertions.In preferred embodiments of the present invention, the further modification is performed in a position selected from the group consisting of : substitution in position 99, substitution in position 133 , substitution in position 143 , substitution in position 167, substitution in position 170, substitution in position 194 , insertion between positions 42 and 43 , insertion between positions 129 and 130, insertion between positions 216 and 217 , insertion between 217 and 218, and combinations thereof .In an interesting embodiment of the invention the additional amino acid residue is inserted between position 98 and 99 (BASBPN numbering) .The insertion between position 98 and 99 is preferably selected from the group consisting of ( in BASBPN numbering)X98X{A, T , G, S } , e . g . , X98XA, X98XT, X98XG, X98XS ; 22X98X{D,E,K,R}, e.g., X98XD,X98XE,X98XK,X98XR; X98X{H,V,C,N,Q}, e.g., X98XH,X98XV,X98XC, X98XN,X98XQ; and X98X{F, I,L,M,P,W,Y} , e.g., X98XF, X98XI , X98XL, X98XM, X98XP, X98XW, X98XY; preferably X98XA, X98XT, X98XG or X98XS;or more specific for subtilisin 309 and closely related subtilases, such as BAALKP, BLSUBL, and BSKSMK:A98A{A,T,G,S} , e.g., A98AA, A98AT, A98AG, A98AS; A98A{D,E,K,R} , e.g., A98AD, A98AE, A98AK, A98AR;A98A{H,V,C,N,Q} , e.g., A98AH, A98AV, A98AC, A98AN, A98AQ;A98A{F, I,L,M,P,W,Y}, e.g. , A98AF, A98AI , A98AL, A98AM, A98AP, A98AW,A98AY; preferably A98AA, A98AT, A98AG or A98AS.Furthermore, it is presently preferred that the insertion between position 98 and 99 is combined with a further modification, namely substitution of an amino acid residue in the positions 133 and 143, as well as substitution of an amino acid residue in the positions 167, 170 and 194.The substitutions (in addition to the insertion between position 98 and 99) in positions 133 and 134, respectively, are preferably selected from the group consisting ofX133{A,T,G,S}, e.g., X133A, X133T,X133G, X133S; X133{D,E,K,R}, e.g., X133D, X133E, X133K, X133R; X133{H,V,C,N,Q}, e.g., X133H, X133V, X133C, X133N, X133Q; X133{F,I,L,M,P,W,Y}, e.g., X133F, X133I ,X133L, X133M, X133P,X133W, X133Y;X143{A,T,G,S}, e.g., X143A, X143T, X143G,X143S ; X143{D,E,K,R} , e.g., X143D,X143E, X143K,X143R; 23X143{H,V,C,N,Q}, e.g., X143H, X143V, X143C,X143N, X143Q; and X143{F,I,L,M,P,W,Y} , e.g., X143F, X143I , X143L, X143M, X143P, X143W, X143Y.In a preferred embodiment the substitution in position 133 is selected from the group consisting of X133 {D, E, K, R} , preferably X133D or X133E, in particular X133E.In another preferred embodiment the substitution in position 143 is selected from the group consisting of X143 {D, E, K, R} , preferably X143K or X143R, in particular X143K.An example of a preferred variant is a subtilase variant comprising the following insertions and substitutions: X98XS+X133E+X143K. A particular preferred variant is a savinase ® variant comprising the following insertions and substitutions A98AS+A133E+T143K.Moreover, the substitutions (in addition to the insertion between position 98 and 99) in positions 167, 170 and 134, respectively, are preferably selected from the group consisting ofX167{A,T,G,S}, e.g., X167A, X167T, X167G, X167S ; X167{D,E,K,R}, e.g., X167D, X167E, X167K, X167R;X167{H,V,C,N,Q} , e.g., X167H, X167V,X167C, X167N, X167Q; X167{F,I,L,M,P,W,Y} , e.g., X167F, X167I ,X167L, X167M, X167P, X167W, X167Y;X170{A,T,G,S} , e.g., X170A, X170T, X170G, X170S ; X170{D,E,K,R} , e.g., X170D,X170E,X170K,X170R; X170{H,V,C,N,Q} , e.g., X170H, X170V, X170C, X170N, X170Q; 24 X170{F,I,L,M,P,W,Y}, e.g., X170F, X170I , X170L,X170M, X170P,X170W, X170Y;X194{A,T,G,S} , e.g., X194A, X194T, X194G, X194S; X194{D,E,K,R} , e.g., X194D, X194E, X194K, X194R;X194{H,V,C,N,Q}, e.g., X194H, X194V, X194C, X194N, X194Q; and X194{F,I,L,M,P,W,Y}, e.g., X194F, X194I , X194L, X194M, X194P, X194W, X194Y.In a preferred embodiment the substitution in position 167 is selected from the group consisting of X167 {A, T, G, S} , in particular X167A; the substitution in position 170 is selected from the group consisting of X170 {A, T, G, S} , in particular X170S; and the substitution in position 194 is selected from the group consisting of X194 {F, I , L, M, P, , Y} , in particular X194P.An example of a preferred variant is a subtilase variant comprising the following insertions and substitutions: X98XT+X167A+X170S+X194P. A particular preferred variant is a savinase® variant comprising the following insertions and substitutions : A98AT+Y167A+R170S+A194P .In a further interesting embodiment of the invention the additional amino acid residue is inserted between position 99 and 100 (BASBPN numbering) .The insertion between position 99 and 100 is preferably selected from the group consisting of (in BASBPN numbering)X99X{A,T,G,S} , e.g., X99XA, X99XT, X99XG, X99XS ; X99X{D,E,K,R} , e.g., X99XD, X99XE, X99XK, X99XR; X99X{H,V,C,N,Q}, e.g., X99XH, X99XV, X99XC, X99XN, X99XQ; and 25X99X{F,I,L,M,P,W,Y}, e.g., X99XF, X99XI , X99XL, X99XM, X99XP, X99XW, X99XY; preferably X99X{D, E, K, R} , in particular X99XD or X99XE;or more specific for subtilisin 309 and closely related subtilases, such as BAALKP, BLSUBL, and BSKSMK:S99S{A,T,G,S}, e.g., S99SA, S99ST, S99SG, S99SS; S99S{D,E,K,R} , e.g., S99SD, S99SE, S99SK, S99SR; S99S{H,V,C,N,Q}, e.g., S99SH, S99SV, S99SC, S99SN, S99SQ; S99S{F,I,L,M,P,W,Y}, e.g. , S99SF, S99SI , S99SL, S99SM, S99SP, S99SW, S99SY; preferably S99S{D, E, K, R} , in particular S99SD or S99SE.With respect to insertions between position 99 and 100, it is - in one interesting embodiment of the present invention - preferred that the insertion is combined with a substitution in position 99. Thus, in addition to the contemplated insertions mentioned above, the following substitutions in position 99 are considered relevant :X99{A,T,G,S}, e.g., X99A, X99T, X99G, X99S; X99{D,E,K,R}, e.g., X99D,X99E, X99K, X99R; X99{H,V,C,N,Q}, e.g., X99H,X99V, X99C, X99N, X99Q; and X99{F,I,L,M,P,W,Y}, e.g., X99F, X99I , X99L, X99M, X99P, X99W, X99Y.In a preferred embodiment the substitution in position 99 is selected from the group consisting of X99 {A, T, G, S} , in particular X99A or X99T.An example of a preferred variant is a subtilase variant comprising the following insertions and substitutions:X99XD+X99A or X99XR+X99T . A particular preferred variant is a 26 savinase® variant comprising the following insertions and substitutions: S99SD+S99A or S99SR+S99T.With respect to insertions between position 99 and 100, it is - in another interesting embodiment of the present invention - preferred that the insertion is combined with a further insertion of at least one amino acid residue between positions216 and 217. Thus, in addition to the contemplated insertions mentioned above, the following insertions between positions 216 and 217 are considered relevant:X216X{A,T,G,S} , e.g., X216XA,X216XT,X216XG,X216XS ; X216X{D,E,K,R} , e.g., X216XD, X216XE, X216XK, X216XR; X216X{H,V,C,N,Q} , e.g., X216XH, X216XV, X216XC, X216XN, X216XQ; and X216X{F,I,L,M,P,W,Y}, e.g., X216XF, X216XI , X216XL, X216XM, X216XP, X216XW,X216XY.In a preferred embodiment the insertion between positions 216 and 217 is selected from the group consisting of X216X{F, I , L, M, P,W,Y} in particular X216XP.Examples of preferred variants are subtilase variants comprising the following insertions and substitutions: X99XD+X99A4-X216XP as well as X99XD+X99A+X216XDP. Particular preferred variants are savinase® variants comprising the following insertions and substitutions: S99SD+S99A+S216SP as well as S99SD+S99A+S216SDP.With respect to insertions between position 99 and 100, it is - in still another interesting eπibodiment of the present invention - preferred that the insertion is coπtbined with a further insertion of at least one amino acid residue between positions217 and 218. Thus, in addition to the contemplated insertions 27 mentioned above, the following insertions between positions 217 and 218 are considered relevant:X217X{A,T,G,S}, e.g., X217XA,X217XT,X217XG, X217XS ; X217X{D,E,K,R} , e.g., X217XD, X217XE, X217XK, X217XR;X217X{H,V,C,N,Q}, e.g., X217XH, X217XV, X217XC, X217XN, X217XQ; and X217X{F,I,L,M,P,W,Y}, e.g., X217XF,X217XI , X217XL, X217XM, X217XP, X217XW,X217XY.In a preferred embodiment the insertion between positions 217 and 218 is selected from the group consisting of X217X{F, I , L, M, P,W,Y} in particular X217XP.Examples of preferred variants are subtilase variants comprising the following insertions and substitutions: X99XD+X99A+X217XP as well as X99XD+X217XP. Particular preferred variants are savinase® variants comprising the following insertions and substitutions: S99SD+S99A+L217LP as well as S99SD+L217P.With respect to insertions between position 99 and 100, it is - in a further interesting embodiment of the present invention - preferred that the insertion is combined with a further insertion of at least one amino acid residue between positions 42 and 43. Thus, in addition to the contemplated insertions mentioned above, the following insertions between positions 42 and 43 are considered relevant:X42X{A,T,G,S} , e.g., X42XA,X42XT,X42XG, X42XS ; X42X{D,E,K,R}, e.g., X42XD,X42XE,X42XK, X42XR; X42X{H,V,C,N,Q} , e.g., X42XH, X42XV, X42XC, X42XN, X42XQ; and X42X{F,I,L,M,P,W,Y}, e.g., X42XF, X42XI , X42XL, X42XM, X42XP, X42XW,X42XY. 28In a preferred embodiment the insertion between positions 42 and 43 is selected from the group consisting of X42X{H, V, C,N, Q} in particular X42XN.Examples of preferred variants are subtilase variants comprising the following insertions and substitutions: X99XD+X42XN as well as X99XD+X99A+X42XN. Particular preferred variants are savinase® variants comprising the following insertions and substitutions: S99SD+D42DN as well as S99SD+S99A+D42DN.With respect to insertions between position 99 and 100, it is - in a still further interesting embodiment of the present invention - preferred that the insertion is combined with a further insertion of at least one amino acid residue between positions 129 and 130. Thus, in addition to the contemplated insertions mentioned above, the following insertions between positions 129 and 130 are considered relevant:X129X{A,T,G,S} , e.g., X129XA,X129XT, X129XG, X129XS; X129X{D,E,K,R} , e.g., X129XD,X129XE, X129XK, X129XR; X129X{H,V,C,N,Q} , e.g., X129XH, X129XV, X129XC, X129XN, X129XQ; and X129X{F,I,L,M,P,W,Y}, e.g., X129XF, X129XI , X129XL, X129XM, X129XP, X129XW,X129XY.In a preferred eπibodiment the insertion between positions 129 and 130 is selected from the group consisting of X129X{D, E, K, R} 29Examples of preferred variants are subtilase variants comprising the following insertions and substitutions: X99XD+X129XD as well as X99XD+X99A+X129XD. Particular preferred variants are savinase® variants comprising the following insertions and substitutions: S99SD+P129PD as well as S99SD+S99A+P129PD.It is well known in the art that a so-called conservative substitution of one amino acid residue to a similar amino acid residue is expected to produce only a minor change in the characteristic of the enzyme.Table I below list groups of conservative amino acid substitutions . 30Table IConservative amino acid substitutionsCommon Property Amino AcidBasic (positive charge) K = lysine H = histidineAcidic (negative charge) E = glutamic acid D = aspartic acidPolar Q = glutamine N = asparagineHydrophobic L = leucine I = isoleucineV = valineM = methionineAromatic F = phenylalanine W = tryptophanY = tyrosineSmall G = glycine A = alanine S = serineT = threonineAccording to this principle subtilase variants comprising conservative substitutions are expected to exhibit characteristics that are not drastically different from each other.Based on the disclosed and/or exemplified subtilase variants herein, it is routine work for a person skilled in the art to identify suitable conservative modification (s) to these variants in order to obtain other subtilase variants exhibiting similarly improved wash-performance . 31It is preferred that the parent subtilase belongs to the subgroups I-Sl and I-S2, especially subgroup I-S2, both for isolating enzymes from nature or from the artificial creation of diversity, and for designing and producing variants from a parent subtilase.In relation to variants from subgroup I-Sl, it is preferred to select a parent subtilase from the group consisting of BSS168 (BSSAS, BSAPRJ, BSAPRN, BMSAMP) , BASBPN, BSSDY, BLSCAR (BLKERA, BLSCA1, BLSCA2, BLSCA3 ) , BSSPRC, and BSSPRD, or functional variants thereof having retained the characteristic of sub-group I-Sl.In relation to variants from subgroup I-S2 it is preferred to select a parent subtilase from the group consisting of BSAPRQ, BLS147 (BSAPRM, BAH101) , BLSAVI (BSKSMK, BAALKP, BLSUBL) , BYSYAB, BAPB92, TVTHER, and BSAPRS, or functional variants thereof having retained the characteristic of sub-group I-S2.In particular, the parent subtilase is BLSAVI (Savinase®, NOVO NORDISK A/S) , and a preferred subtilase variant of the invention is accordingly a variant of Savinase®. Thus, particular interesting variants are savinase® variants, i.e. BLSAVI variants, wherein

1. Ser has been inserted between positions 98 and 99, Ala in position 133 has been substituted with Glu, and Thr in position 143 has been substituted with Lys (BASBPN number- ing) ; or 32

2. Asp has been inserted between positions 99 and 100 and Ser in position 99 has been substituted with Ala (BASBPN numbering) ; or

3. Thr has been inserted between positions 98 and 99, Tyr in position 167 has been substituted with Ala, Arg in position 170 has been substituted with Ser, and Ala in position 194 has been substituted with Pro (BASBPN numbering) ; or

4. Asp has been inserted between positions 99 and 100, Ser in position 99 has been substituted with Ala, and Pro has been inserted between positions 217 and 218 (BASBPN numbering) .

5. Asp has been inserted between positions 99 and 100, Ser in position 99 has been substituted with Ala, and Pro has been inserted between positions 216 and 217 (BASBPN numbering) .

6. Asp has been inserted between positions 99 and 100, Ser in position 99 has been substituted with Ala, and Asp-Pro has been inserted between positions 216 and 217 (BASBPN num- bering) .

7. Asp has been inserted between positions 99 and 100, Ser in position 99 has been substituted with Ala, and Asp has been inserted between positions 129 and 130 (BASBPN numbering) . 8. Asp has been inserted between positions 99 and 100, and

Asn has been inserted between positions 42 and 43 (BASBPN numbering) .

9. Asp has been inserted between positions 99 and 100, Ser in position 99 has been substituted with Ala, and Asn has been inserted between positions 42 and 43 (BASBPN nuπiber- ing) . 33

10. Arg has been inserted between posiions 99 and 100, and Ser in position 99 has been substituted with Thr.

11. Asp has been inserted between positions 99 and 100, Ser in position 99 has been substituted with Ala, and Pro in position 131 has been substituted with Thr.

The present invention also encompass use of any of the above mentioned subtilase variants in combination with any other modification to the amino acid sequence thereof. Especially combina- tions with other modifications known in the art to provide improved properties to the enzyme are envisaged. The art describes a number of subtilase variants with different improved properties and a number of those are mentioned in the "Background of the invention" section herein ( vide supra) . Those references are disclosed here as references to identify a subtilase variant, which advantageously can be combined with a subtilase variant described herein.

Such combinations comprise the positions: 222 (improves oxida- tion stability) , 218 (improves thermal stability) , substitutions in the Ca-binding sites stabilizing the enzyme, e.g. position 76, and many other apparent from the prior art.

In further embodiments a subtilase variant described herein may advantageously be combined with one or more modification (s) in any of the positions:

27, 36, 56, 76, 87, 97, 101, 103, 104, 120, 123, 159, 167, 170, 206, 218, 222, 224, 232, 235, 236, 245, 248, 252 and 274.

Specifically the following BLSAVI, BLSUBL, BSKSMK, and BAALKP variants are considered appropriate for combination: 34

K27R, *36D, S56P, N76D, S87N, G97N, S101G, S103A, V104A, V104I, V104N, V104Y, H120D, N123S, G159D, Y167, R170, Q206E, N218S, M222S, M222A, T224S, A232V, K235L, Q236H, Q245R, N248D, N252K and T274A.

Furthermore variants comprising any of the variants S101G+V104N, S87N+S101G+V104N, K27R+V104Y+N123S+T274A, N76D+S103A+V104I or N76D+V104A or other combinations of these mutations (V104N, S101G, K27R, V104Y, N123S, T274A, N76D, V104A) or

S101G+S103A+V104I+G159D-.A232V+Q236H+Q245R+N248D+N252K in combination with any one or more of the modification (s) mentioned above exhibit improved properties.

Moreover, subtilase variants of the main aspect (s) of the invention are preferably combined with one or more modification (s) in any of the positions 129, 131 and 194, preferably as 129K, 131H and 194P modifications, and most preferably as P129K, P131H and A194P modifications. Any of those modification (s) are expected to provide a higher expression level of the subtilase variant in the production thereof.

As mentioned above, the variants disclosed herein are only inhibited by trypsin inhibitor type IV- 0 to a limited extent and, consequently, they exhibit excellent wash performance on egg stains. Therefore, in order to enable the skilled person - at an early stage of his development work - to select effective and preferred variants for this purpose, the present inventors have provided a suitable preliminary test, which can easily be carried out by the skilled person in order to initially assess the performance of the variant in question. 35

Thus, the "Ovo-inhibition Assay" disclosed in Example 4 herein may be employed to initially assess the potential of a selected variant. In other words, the "Ovo-inhibition Assay" may be employed to assess whether a selected variant will be inhibited, and to what extent, by the trypsin inhibitor type IV- 0. Using this test, the suitability of a selected variant to remove egg stains can be assessed, the rationale being that if a selected variant is strongly inhibited by trypsin inhibitor type IV- 0, it is normally not necessary to carry out further test experiments.

Therefore, a variant which is particular interesting for the use described herein, is a variant which - when tested in the "Ovo- inhibition Assay" described in Example 4 herein - has a Residual Activity of at least 10%, e.g. at least 15%, such as at least 20%, preferably at least 25%, such as at least 30%, more preferably at least 35%. In a particular interesting embodiment of the invention, the variant has a Residual Activity of at least 40%, such as at least 45%, e.g. at least 50%, preferably at least 55%, such as at least 60%, more preferably at least 65%, such as at least 70%, even more preferably at least 75%, such as at least 80%, e.g. at least 90%, when tested in the "Ovo-inhibition Assay" described in Example 4 herein.

Evidently, it is preferred that the variant of the invention fulfils the above criteria on at least the stated lowest level, more preferably at the stated intermediate level and most preferably on the stated highest level.

Alternatively, or in addition to the above-mentioned assay, the suitability of a selected variant may be tested in the "Model Detergent Wash Performance Test" disclosed in Example 3 herein. The "Model Detergent Wash Perfomance Test" may be employed to 36 assess the ability of a variant, when incorporated in a standard detergent composition, to remove egg stains from a standard textile as compared to a reference system, namely the parent subtilase (incorporated in the same model detergent system and tested under identical conditions) . Using this test, the suitability of a selected variant to remove egg stains can be initially investigated, the rationale being that if a selected variant does not show a significant improvement in the test compared to the parent subtilase, it is normally not necessary to carry out further test experiments.

Therefore, variants which are particular interesting for the use described herein, are such variants which, when tested in a model detergent composition comprising

6.2% LAS (Nansa 80S)

2% Sodium salt of C₁₆-C₁₈ fatty acid

4% Non-ionic surfactant (Plurafax LF404]

22% Zeolite P

10.5% Na₂C0₃

4% Na₂Si₂0₅

2% Carboxymethylcellulose (CMC)

6.8% Acrylate liquid CP5 40% 20% Sodium perborate (empirical formula NaB0₂.H₂0₂) 0.2% EDTA

21% Na₂S0₄

Water (balance)

as described in the "Model Detergent Wash Performance Test" disclosed in Example 3 herein, shows an improved wash performance on egg stains as compared to the parent subtilase tested under identical conditions. 37

The improvement in the wash performance may be quantified by employing the so-called "Performance Factor" defined in Example 3, herein.

In a very interesting embodiment of the invention, the variant of the invention, when tested in the "Wash Performance Test" has a Performance Factor of at least 1, such as at least 1.5, e.g. at least 2, preferably at least 2.5, such as at least 3, e.g. at least 3.5, in particular at least 4, such as at least 4.5, e.g. at least 5.

Evidently, it is preferred that the variant of the invention fulfils the above criteria on at least the stated lowest level, more preferably at the stated intermediate level and most preferably on the stated highest level .

As indicated above, the present invention also provides novel subtilase variants. It will be understood that details and par- ticulars concerning the novel subtilase variant aspects of the invention will be the same or analogous to the details and particulars of the variants discussed above in connection with the use aspect of the invention. This means that whenever appropriate, the statements concerning the use (e.g. preferred inser- tions and substitutions, etc.) discussed in detail herein, apply mutatis mutandis to the novel subtilase variants according to the invention as well as to the method aspect and the cleaning and detergent composition aspect of the invention.

PRODUCING A SUBTILASE VARIANT

Many methods for cloning a subtilase and for introducing insertions into genes (e.g. subtilase genes) are well known in the 38 art, cf. the references cited in the "BACKGROUND OF THE INVENTION" section.

In general standard procedures for cloning of genes and intro- ducing insertions (random and/or site directed) into said genes may be used in order to obtain a subtilase variant of the invention. For further description of suitable techniques reference is made to Examples herein ( vide infra) and (Sambrook et al . (1989) Molecular cloning: A laboratory manual, Cold Spring Har- bor lab., Cold Spring Harbor, NY; Ausubel, F. M. et al . (eds.) "Current protocols in Molecular Biology". John Wiley and Sons, 1995; Harwood, C. R. , and Cutting, S. M. (eds.) "Molecular Biological Methods for Bacillus". John Wiley and Sons, 1990); and WO 96/34946.

Further, a subtilase variant may be constructed by standard techniques for artificial creation of diversity, such as by DNA shuffling of different subtilase genes (WO 95/22625; Stemmer WPC, Nature 370:389-91 (1994)). DNA shuffling of e.g. the gene encoding Savinase® with one or more partial subtilase sequences identified in nature to comprise an active site (b) loop regions longer than the active site (b) loop of Savinase®, will after subsequent screening for improved wash performance variants, provide subtilase variants suitable for the purposes described herein.

EXPRESSION VECTORS

A recombinant expression vector comprising a DNA construct encoding the enzyme of the invention may be any vector which may conveniently be subjected to recombinant DNA procedures.

The choice of vector will often depend on the host cell into which it is to be introduced. Thus, the vector may be an 39 autonomously replicating vector, i.e. a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid.

Alternatively, the vector may be one that on introduction into a host cell is integrated into the host cell genome in part or in its entirety and replicated together with the chromosome (s) into which it has been integrated.

The vector is preferably an expression vector in which the DNA sequence encoding the enzyme of the invention is operably linked to additional segments required for transcription of the DNA. In general, the expression vector is derived from plasmid or viral DNA, or may contain elements of both. The term, "operably linked" indicates that the segments are arranged so that they function in concert for their intended purposes, e.g. transcription initiates in a promoter and proceeds through the DNA sequence coding for the enzyme .

The promoter may be any DNA sequence which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell .

Examples of suitable promoters for use in bacterial host cells include the promoter of the Bacillus stearothermophilus maltogenic amylase gene, the Bacillus licheniformis alpha- amylase gene, the Bacillus a yloliquefaciens alpha-amylase gene, the Bacillus subtilis alkaline protease gen, or the Bacillus pumilus xylosidase gene, or the phage Laπibda P_R or P_L promoters or the E. coli lac, trp or tac promoters. 40

The DNA sequence encoding the enzyme of the invention may also, if necessary, be operably connected to a suitable terminator.

The recombinant vector of the invention may further comprise a DNA sequence enabling the vector to replicate in the host cell in question.

The vector may also comprise a selectable marker, e.g. a gene the product of which complements a defect in the host cell, or a gene encoding resistance to e.g. antibiotics like kanamycin, chloramphenicol, erythromycin, tetracycline, spectinomycine, or the like, or resistance to heavy metals or herbicides.

To direct an enzyme of the present invention into the secretory pathway of the host cells, a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) may be provided in the recombinant vector. The secretory signal sequence is joined to the DNA sequence encoding the enzyme in the correct reading frame. Secretory signal sequences are commonly positioned 5' to the DNA sequence encoding the enzyme. The secretory signal sequence may be that normally associated with the enzyme or may be from a gene encoding another secreted protein.

The procedures used to ligate the DNA sequences coding for the present enzyme, the promoter and optionally the terminator and/or secretory signal sequence, respectively, or to assemble these sequences by suitable PCR amplification schemes, and to insert them into suitable vectors containing the information necessary for replication or integration, are well known to persons skilled in the art (cf., for instance, Sambrook et al . , op. cit . ) . 41

HOST CELL

The DNA sequence encoding the present enzyme introduced into the host cell may be either homologous or heterologous to the host in question. If homologous to the host cell, i.e. produced by the host cell in nature, it will typically be operably connected to another promoter sequence or, if applicable, another secretory signal sequence and/or terminator sequence than in its natural environment. The term "homologous" is intended to include a DNA sequence encoding an enzyme native to the host organism in question. The term "heterologous" is intended to include a DNA sequence not expressed by the host cell in nature. Thus, the DNA sequence may be from another organism, or it may be a synthetic sequence.

The host cell into which the DNA construct or the recombinant vector of the invention is introduced may be any cell which is capable of producing the present enzyme and includes bacteria, yeast, fungi and higher eukaryotic cells including plants.

Examples of bacterial host cells which, on cultivation, are capable of producing the enzyme of the invention are gram- positive bacteria such as strains of Bacillus, such as strains of B . subtilis , B . licheniformis, B . lentus , B . brevis, B . stearothermophilus , B . alkalophilus, B . amyloliquefaciens , B . coagulans, B . circulans, B . lautus, B . megatherium or B . thuringiensis, or strains of Streptomyces, such as S. lividans or S . murinus, or gram-negative bacteria such as Echerichia coli .

The transformation of the bacteria may be effected by protoplast transformation, electroporation, conjugation, or by using 42 competent cells in a manner known per se (cf. Sambrook et al . , supra) .

When expressing the enzyme in bacteria such as E. coli , the enzyme may be retained in the cytoplasm, typically as insoluble granules (known as inclusion bodies) , or may be directed to the periplasmic space by a bacterial secretion sequence. In the former case, the cells are lysed and the granules are recovered and denatured after which the enzyme is refolded by diluting the denaturing agent. In the latter case, the enzyme may be recovered from the periplasmic space by disrupting the cells, e.g. by sonication or osmotic shock, to release the contents of the periplasmic space and recovering the enzyme.

When expressing the enzyme in gram-positive bacteria such as

Bacillus or Streptomyces strains, the enzyme may be retained in the cytoplasm, or may be directed to the extracellular medium by a bacterial secretion sequence. In the latter case, the enzyme may be recovered from the medium as described below.

METHOD FOR PRODUCING A SUBTILASE VARIANT

The present invention provides a method of producing an isolated enzyme according to the invention, wherein a suitable host cell, which has been transformed with a DNA sequence encoding the enzyme, is cultured under conditions permitting the production of the enzyme, and the resulting enzyme is recovered from the culture .

When an expression vector comprising a DNA sequence encoding the enzyme is transformed into a heterologous host cell it is possible to enable heterologous recombinant production of the enzyme of the invention. 43

Thereby it is possible to make a highly purified subtilase composition, characterized in being free from homologous impurities .

In this context homologous impurities means any impurities (e.g, other polypeptides than the enzyme of the invention) which originate from the homologous cell where the enzyme of the invention is originally obtained from.

The medium used to culture the transformed host cells may be any conventional medium suitable for growing the host cells in question. The expressed subtilase may conveniently be secreted into the culture medium and may be recovered therefrom by well- known procedures including separating the cells from the medium by centrifugation or filtration, precipitating proteinaceous components of the medium by means of a salt such as ammonium sulfate, followed by chromatographic procedures such as ion exchange chromatography, affinity chromatography, or the like.

CLEANING AND DETERGENT COMPOSITIONS

In general, cleaning and detergent compositions are well described in the art and reference is made to WO 96/34946; WO 97/07202; WO 95/30011 for further description of suitable cleaning and detergent compositions.

Furthermore the examples herein demonstrate the improvements in wash performance on egg stains for a number of subtilase variants .

Detergent Compositions 44

The subtilase variant may be added to and thus become a component of a detergent composition.

The detergent composition of the invention may for example be formulated as a hand or machine laundry detergent composition including a laundry additive composition suitable for pre- treatment of stained fabrics and a rinse added fabric softener composition, or be formulated as a detergent composition for use in general household hard surface cleaning operations, or be formulated for hand or machine dishwashing operations.

In a specific aspect, the invention provides a detergent additive comprising a subtilase enzyme of the invention. The detergent additive as well as the detergent composition may comprise one or more other enzymes such as another protease, a lipase, a cutinase, an amylase, a carbohydrase, a cellulase, a pectinase, a mannanase, an arabinase, a galactanase, a xylanase, an oxi- dase, e.g., a laccase, and/or a peroxidase.

In general the properties of the chosen enzyme (s) should be compatible with the selected detergent, (i.e. pH-optimum, compatibility with other enzymatic and non-enzymatic ingredients, etc.), and the enzyme (s) should be present in effective amounts.

Proteases : Suitable proteases include those of animal, vegetable or microbial origin. Microbial origin is preferred. Chemically modified or protein engineered mutants are included. The protease may be a serine protease or a metallo protease, preferably an alkaline microbial protease or a trypsin- like protease. Examples of alkaline proteases are subtilisins, especially those derived from Bacillus , e.g., subtilisin Novo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 and 45 subtilisin 168 (described in WO 89/06279) . Examples of trypsin- like proteases are trypsin (e.g. of porcine or bovine origin) and the Fusarium protease described in WO 89/06270 and WO 94/25583.

Examples of useful proteases are the variants described in WO 92/19729, WO 98/20115, WO 98/20116, and WO 98/34946, especially the variants with substitutions in one or more of the following positions: 27, 36, 57, 76, 87, 97, 101, 104, 120, 123, 167, 170, 194, 206, 218, 222, 224, 235 and 274.

Preferred commercially available protease enzymes include Alcalase™, Savinase™, Primase™, Duralase™, Esperase™, and Kannase™ (Novo Nordisk A/S) , Maxatase™, Maxacal™, Maxapem™, Properase™, Purafect™, Purafect OxP™, FN2™, and FN3™ (Genencor International Inc.) .

Lipases : Suitable lipases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful lipases include lipases from Humicola (synonym Thermomyces) , e.g. from H. lanuginosa ( T. lanuginosus) as described in EP 258 068 and EP 305 216 or from H. insolens as described in WO 96/13580, a Pseudomonas lipase, e.g. from P. alcaligenes or P. pseudoalcaligenes (EP 218 272), P. cepacia (EP 331 376), P. stutzeri (GB 1,372,034), P. fluorescens, Pseudomonas sp . strain SD 705 (WO 95/06720 and WO 96/27002) , P. wisconsinensis (WO 96/12012) , a Bacillus lipase, e.g. from B . subtili s (Dartois et al . (1993), Biochemica et Biophysica Acta, 1131, 253-360), B . stearothermophilus (JP 64/744992) or B . pumilus (WO 91/16422) . 46

Other examples are lipase variants such as those described in WO 92/05249, WO 94/01541, EP 407 225, EP 260 105, WO 95/35381, WO 96/00292, WO 95/30744, WO 94/25578, WO 95/14783, WO 95/22615, WO 97/04079 and WO 97/07202.

Preferred commercially available lipase enzymes include Lipolase™ and Lipolase Ultra™ (Novo Nordisk A/S) .

Amylases : Suitable amylases (α and/or β) include those of bac- terial or fungal origin. Chemically modified or protein engineered mutants are included. Amylases include, for example, -amylases obtained from Bacillus, e.g. a special strain of B . licheniformis, described in more detail in GB 1,296,839.

Examples of useful amylases are the variants described in WO

94/02597, WO 94/18314, WO 96/23873, and WO 97/43424, especially the variants with substitutions in one or more of the following positions: 15, 23, 105, 106, 124, 128, 133, 154, 156, 181, 188, 190, 197, 202, 208, 209, 243, 264, 304, 305, 391, 408, and 444.

Commercially available amylases are Duramyl™, Termamyl™, Fun- gamyl™ and BAN™ (Novo Nordisk A/S) , Rapidase™ and Purastar™ (from Genencor International Inc.) .

Cellulases : Suitable cellulases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Suitable cellulases include cellulases from the genera Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, e.g. the fungal cellulases produced from Humicola insolens, Myceliophthora thermophila and Fusarium oxysporum disclosed in US 4,435,307, US 5,648,263, US 5,691,178, US 5,776,757 and WO 89/09259. 47

Especially suitable cellulases are the alkaline or neutral cellulases having colour care benefits. Examples of such cellulases are cellulases described in EP 0 495 257, EP 0 531 372, WO 96/11262, WO 96/29397, WO 98/08940. Other examples are cellulase variants such as those described in WO 94/07998, EP 0 531 315, US 5,457,046, US 5,686,593, US 5,763,254, WO 95/24471, WO 98/12307 and PCT/DK98/00299.

Commercially available cellulases include Celluzyme™, and Carezyme™ (Novo Nordisk A/S) , Clazinase™, and Puradax HA™ (Genencor International Inc.), and KAC-500(B)™ (Kao Corporation) .

Peroxidases/Oxidases : Suitable peroxidases/oxidases include those of plant, bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful peroxidases include peroxidases from Coprinus , e.g. from C. cinereus, and variants thereof as those described in WO 93/24618, WO 95/10602, and WO 98/15257.

Commercially available peroxidases include Guardzyme™ (Novo Nordisk A/S) .

The detergent enzyme (s) may be included in a detergent composition by adding separate additives containing one or more enzymes, or by adding a combined additive comprising all of these enzymes. A detergent additive of the invention, i.e. a separate additive or a combined additive, can be formulated e.g. as a granulate, a liquid, a slurry, etc. Preferred detergent additive formulations are granulates, in particular non-dusting granulates, liquids, in particular stabilized liquids, or slurries. 48

Non-dusting granulates may be produced, e.g., as disclosed in US 4,106,991 and 4,661,452 and may optionally be coated by methods known in the art. Examples of waxy coating materials are poly (ethylene oxide) products (polyethyleneglycol, PEG) with mean molar weights of 1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains from 12 to 20 carbon atoms and in which there are 15 to 80 ethylene oxide units; fatty al- cohols; fatty acids; and mono- and di- and triglycerides of fatty acids. Examples of film-forming coating materials suitable for application by fluid bed techniques are given in GB 1483591. Liquid enzyme preparations may, for instance, be stabilized by adding a polyol such as propylene glycol, a sugar or sugar alco- hol, lactic acid or boric acid according to established methods. Protected enzymes may be prepared according to the method disclosed in EP 238,216.

The detergent composition of the invention may be in any conven- ient form, e.g., a bar, a tablet, a powder, a granule, a paste or a liquid. A liquid detergent may be aqueous, typically containing up to 70% water and 0-30% organic solvent, or non- aqueous .

The detergent composition typically comprises one or more surfactants, which may be non- ionic including semi -polar and/or anionic and/or cationic and/or zwitterionic. The surfactants are typically present at a level of from 0.1% to 60% by weight. When included therein the detergent will usually contain from about 1% to about 40% of an anionic surfactant such as linear alkylbenzenesulfonate, alpha-olefinsulfonate, alkyl sulfate (fatty alcohol sulfate) , alcohol ethoxysulfate, secondary al- 4 9 kanesulfonate, alpha-sulfo fatty acid methyl ester, alkyl- or alkenylsuccmic acid or soap.

When included therein the detergent will usually contain from about 0.2% to about 40% of a non-ionic surfactant such as alcohol ethoxylate, nonylphenol ethoxylate, alkylpolyglycoside, al- kyldimethylamineoxide, ethoxylated fatty acid monoethanolamide, fatty acid monoethanolamide, polyhydroxy alkyl fatty acid amide, or N-acyl N-alkyl derivatives of glucosamine ( "glucamides" ) .

The detergent may contain 0-65% of a detergent builder or com- plexing agent such as zeolite, diphosphate, triphosphate, phosphonate, carbonate, citrate, nitrilotriacetic acid, ethylenedia- minetetraacetic acid, diethylenetriaminepentaacetic acid, alkyl - or alkenylsuccmic acid, soluble silicates or layered silicates (e.g. SKS-6 from Hoechst) .

The detergent may comprise one or more polymers. Examples are carboxymethylcellulose, poly (vinylpyrrolidone) , poly (ethylene glycol), poly(vinyl alcohol), poly (vinylpyridine-N-oxide) , poly (vinylimidazole) , polycarboxylates such as polyacrylates, maleic/acrylic acid copolymers and lauryl methacrylate/acrylic acid copolymers.

The detergent may contain a bleaching system which may comprise a H₂0₂ source such as perborate or percarbonate which may be combined with a peracid-forming bleach activator such as tetraace- tylethylenediamine or nonanoyloxybenzenesulfonate . Alternatively, the bleaching system may comprise peroxyacids of e.g. the amide, imide, or sulfone type. 50

The enzyme (s) of the detergent composition of the invention may be stabilized using conventional stabilizing agents, e.g., a polyol such as propylene glycol or glycerol, a sugar or sugar alcohol, lactic acid, boric acid, or a boric acid derivative, e.g., an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid, and the composition may be formulated as described in e.g. WO 92/19709 and WO 92/19708.

The detergent may also contain other conventional detergent ingredients such as e.g. fabric conditioners including clays, foam boosters, suds suppressors, anti -corrosion agents, soil- suspending agents, anti -soil redeposition agents, dyes, bacte- ricides, optical brighteners, hydrotropes, tarnish inhibitors, or perfumes.

It is at present contemplated that in the detergent compositions any enzyme, in particular the enzyme of the invention, may be added in an amount corresponding to 0.01-100 mg of enzyme pro- tein per liter of wash liquor, preferably 0.05-5 mg of enzyme protein per liter of wash liquor, in particular 0.1-1 mg of enzyme protein per liter of wash liquor.

The enzyme of the invention may additionally be incorporated in the detergent formulations disclosed in WO 97/07202 which is hereby incorporated as reference.

The invention is described in further detail in the following examples, which are not in any way intended to limit the scope of the invention as claimed. 51

In the detergent compositions, the abbreviated component identifications have the following meanings:

LAS: Sodium linear C₁₂ alkyl benzene sulphonate

TAS : Sodium tallow alkyl sulphate

XYAS : Sodium C_1X - C_lγ alkyl sulfate

SS: Secondary soap surfactant of formula 2 -butyl oc- tanoic acid

25EY: A C₁₂-C₁₅ predominantly linear primary alcohol condensed with an average of Y moles of ethylene oxide

45EY: A C₁₄-C_1S predominantly linear primary alcohol condensed with an average of Y moles of ethylene oxide

XYEZS : C_1X-C_1Y sodium alkyl sulfate condensed with an average of Z moles of ethylene oxide per mole

Nonionic : C₁₃-C₁₅ mixed ethoxylated/propoxylated fatty alcohol with an average degree of ethoxylation of 3.8 and an average degree of propoxylation of 4.5 sold under the tradename Plurafax LF404 by BASF GmbH

CFAA: C₁₂-C₁₄ alkyl N-methyl glucamide

TFAA: Ci₆ ^_ci₈ alkyl N-methyl glucamide

Silicate: Amorphous Sodium Silicate (Si0₂:Na₂0 ratio = 2.0) 52 NaSKS-6: Crystalline layered silicate of formula δ-Na₂Si₂0₅

Carbonate: Anhydrous sodium carbonate

Phosphate: Sodium tripolyphosphate

MA/AA: Copolymer of 1:4 maleic/acrylic acid, average molecular weight about 80,000

Polyacrylate: Polyacrylate homopolymer with an average molecular weight of 8,000 sold under the trade- name PA30 by BASF Gmbh

Zeolite A: Hydrated Sodium Alummosilicate of formula Na₁₂ (Al0₂Si0₂) ₁₂.27H₂0 having a primary particle size in the range from 1 to 10 micrometers

Citrate: Tri-sodium citrate dihydrate

Citric: Citric Acid

Perborate: Anhydrous sodium perborate monohydrate bleach, empirical formula NaB0₂.H₂0₂

PB4 : Anhydrous sodium perborate tetrahydrate

Percarbonate : Anhydrous sodium percarbonate bleach of empirical formula 2Na₂C0₃.3H₂0₂

TAED: Tetraacetyl ethylene diamine

CMC: Sodium carboxymethyl cellulose 53

DETPMP: Diethylene triamine penta (methylene phosphonic acid) , marketed by Monsanto under the Tradename De- quest 2060

PVP: Polyvinylpyrrolidone polymer

EDDS : Ethylenediamine-N, N' -disuccinic acid, [S,S] isomer in the form of the sodium salt

Suds 25% paraffin wax Mpt 50°C, 17% hydrophobic silica, Suppressor: 58% paraffin oil

Granular Suds 12% Silicone/silica, 18% stearyl alcohol, 70%

suppressor: starch in granular form

Sulphate: Anhydrous sodium sulphate

HMWPEO : High molecular weight polyethylene oxide

TAE 25: Tallow alcohol ethoxylate (25)

Detergent Example I A granular fabric cleaning composition in accordance with the invention may be prepared as follows:

Sodium linear C₁₂ alkyl 6.5 benzene sulfonate

Sodium sulfate 15.0

Zeolite A 26.0 54

Sodium nitrilotriacetate 5.0

Enzyme 0.1

PVP 0.5

TAED 3.0

Boric acid 4.0

Perborate 18.0

Phenol sulphonate 0.1

Minors • up to 100%

Detergent Example II

A compact granular fabric cleaning composition (density 800 g/1) in accord with the invention may be prepared as follows:

45AS 8.0 25E3S 2.0

25E5 3.0

25E3 3.0

TFAA 2.5

Zeolite A 17.0 NaSKS-6 12.0

Citric acid 3.0

Carbonate 7.0

MA/AA 5.0

CMC 0.4 Enzyme 0.1

TAED 6.0

Percarbonate 22.0

EDDS 0.3

Granular suds suppressor 3.5 water/minors Up to 100%

Detergent Example III 55

Granular fabric cleaning compositions in accordance with the invention which are especially useful in the laundering of coloured fabrics were prepared as follows:

LAS 10.7 -

TAS 2.4 -

TFAA - 4.0

45AS 3.1 10.0

45E7 4.0 -

25E3S - 3.0

68E11 1.8 -

25E5 - 8.0

Citrate 15.0 7.0

Carbonate - 10.0

Citric acid 2.5 3.0

Zeolite A 32.1 25.0

Na-SKS-6 - 9.0

MA/AA 5.0 5.0

DETPMP 0.2 0.8

Enzyme 0.10 0.05

Silicate 2.5 -

Sulphate 5.2 3.0

PVP 0.5 -

Poly (4-vinylpyridine :) -N- - 0.2

Oxide/copolymer of vi .nyl- imidazole and vinyl - pyrrolidone

Perborate 1.0 -

Phenol sulfonate 0.2 -

Water/Minors Up to 100% 56

Detergent Example IV

Granular fabric cleaning compositions in accordance with the invention which provide "Softening through the wash" capability may be prepared as follows:

45AS - 10 .0

LAS 7 .6 -

68AS 1 .3 -

45E7 4 .0 -

25E3 - 5 .0

Coco-alkyl -dimethyl hydroxy- 1 .4 1 .0 ethyl ammonium chloride

Citrate 5 .0 3 .0

Na-SKS-6 - 11 .0

Zeolite A 15 .0 15 .0

MA/AA 4 , .0 4, .0

DETPMP 0, .4 0, .4

Perborate 15, .0 -

Percarbonate - 15, .0

TAED 5. .0 5. .0

Smectite clay 10. .0 10. .0

HMWPEO - 0. .1

Enzyme 0. .10 0. ,05

Silicate 3. .0 5. ,0

Carbonate 10. .0 10. ,0

Granular suds suppressor 1. .0 4. ,0

CMC 0. 2 0. .1

Water/Minors Up to 100%

Detergent Example V 57

Heavy duty liquid fabric cleaning compositions in accordance with the invention may be prepared as follows:

LAS acid form - 25 .0

Citric acid 5 0 2 .0

25AS acid form 8 0 -

25AE2S acid form 3 0 -

25AE7 8 0 -

CFAA 5 -

DETPMP 1 0 1 0

Fatty acid 8 -

Oleic acid - 1 0

Ethanol 4 0 6 0

Propanediol 2 0 6 0

Enzyme 0 10 0 05

Coco-alkyl dime thyl - 3 0 hydroxy ethyl ammonium chloride

Smectite clay 5.0

PVP 2.0

Water / Minors Up to 100%

Powder automatic dishwash composition I

Nonionic surfactant 0.4 - 2.5%

Sodium metasilicate 0 - 20%

Sodium disilicate 3 - 20%

Sodium triphosphate 20 - 40%

Sodium carbonate 0 - 20%

Sodium perborate 2 - 9% 58

Powder automatic dishwash composition II

Nonionic surfactant 1 - 2% (e.g. alcohol ethoxylate)

Sodium disilicate 2 - 30%

Sodium carbonate 10 - 50%

Sodium phosphonate 0 - 5%

Trisodium citrate dihydrate 9 - 30%

Nitrilotrisodium acetate (NTA) 0 - 20%

Sodium perborate monohydrate 5 - 10%

Tetraacetyl ethylene diamine (TAED) 1 - 2%

Polyacrylate polymer (e.g. maleic acid/acrylic acid co- 6 - 25% polymer)

Enzymes 0.0001 0.1%

Perfume 0.1 - 0.5%

Water 5 - 10

Powder automatic dishwash composition III

Nonionic surfactant 0.5 - 2.0%

Sodium disilicate 25 - 40%

Sodium citrate 30 - 55%

Sodium carbonate 0 - 29%

Sodium bicarbonate 0 - 20%

Sodium perborate monohydrate 0 - 15% 59

Tetraacetyl ethylene diamine (TAED) 0 - 6%

Maleic acid/acrylic 0 - 5% acid copolymer

Clay 1 - 3%

Polyamino acids 0 - 20%

Sodium polyacrylate 0 - 8%

Enzymes 0.0001 - - 0.1%

Powder automatic dishwash composition IV

Nonionic surfactant 1 2%

Zeolite MAP 15 42%

Sodium disilicate 30 34%

Sodium citrate 0 12%

Sodium carbonate 0 20%

Sodium perborate monohydrate 7 15%

Tetraacetyl ethylene diamine (TAED) 0 3%

Polymer 0 4%

Maleic acid/acrylic acid copolymer 0 5%

Organic phosphonate 0 4%

Clay 1 2%

Enzymes 0.0001 - 0.1%

Sodium sulphate Balance

Powder automatic dishwash composition V

60

Powder and liquid dishwash composition with cleaning surfactant system VI

61 oxylation of 9

A blend of C₁₃-C₁₅ ethoxylated alcohols with an average degree of eth0 - 4% oxylation of 30

Sodium disilicate 0 - 33%

Sodium tripolyphosphate 0 - 46%

Sodium citrate 0 - 28%

Citric acid 0 - 29%

Sodium carbonate 0 - 20%

Sodium perborate monohydrate 0 - 11.5%

Tetraacetyl ethylene diamine (TAED) 0 - 4%

Maleic acid/acrylic acid copolymer 0 - 7.5%

Sodium sulphate 0 - 12.5%

Enzymes 0.0001 - 0.1%

Non-aqueous liquid automatic dishwshing composition VII

Liquid nonionic surfactant (e.g. alcohol ethoxylates) 2.0 - 10.0%

Alkali metal silicate 3.0 - 15.0%

Alkali metal phosphate 20.0 - 40.0%

Liquid carrier selected from higher glycols, polyglycols, polyoxides, 25.0 - 45.0% glycolethers

Stabilizer (e.g. a partial ester of phosphoric acid and a C₁₆-C₁₈ alka- 0.5 - 7.0% nol)

Foam suppressor (e.g. silicone) 0 1.5%

Enzymes 0.0001 - 0.1% 62

Non-aqueous liquid dishwashing composition VIII

Liquid nonionic surfactant (e.g. alcohol ethoxylates) 2.0 - 10.0%

Sodium silicate 3.0 - 15.0%

Alkali metal carbonate 7.0 - 20.0%

Sodium citrate 0.0 - 1.5%

Stabilizing system (e.g. mixtures of finely divided silicone and low molecular weight dialkyl polyglycol 0.5 - 7.0% ethers)

Low molecule weight polyacrylate polymer 5.0 - 15.0%

Clay gel thickener (e.g. bentonite) 0.0 - 10.0%

Hydroxypropyl cellulose polymer 0.0 - 0.6%

Enzymes 0.0001 - 0.1%

Liquid carrier selected from higher lycols, polyglycols, polyoxides and Balance glycol ethers

Thixotropic liquid automatic dishwashing composition IX

^Ci₂ ^-Ci₄ fatty acid 0 - 0.5%

Block co-polymer surfactant 1.5 - 15.0%

Sodium citrate 0 - 12%

Sodium tripolyphosphate 0 - 15%

Sodium carbonate 0 - 8%

Aluminium tristearate 0 0.1%

Sodium cumene sulphonate 0 - 1.7%

Polyacrylate thickener 1.32 - 2.5%

Sodium polyacrylate 2.4 - 6.0% 63

Boric acid 0 4.0%

Sodium formate 0 0.45%

Calcium formate 0 0.2%

Sodium n-decydiphenyl oxide disul- phonate 0 4.0%

Monoethanol amine (MEA) 0 1.86%

Sodium hydroxide (50%) 1.9 9.3%

1,2- Propanediol 0 9.4%

Enzymes 0.0001 - 0.1%

Suds suppressor, dye, perfumes, water Balance

Liquid automatic dishwashing composition X

Alcohol ethoxylate 0 - 20%

Fatty acid ester sulphonate 0 - 30%

Sodium dodecyl sulphate 0 - 20%

Alkyl polyglycoside 0 - 21%

Oleic acid 0 - 10%

Sodium disilicate monohydrate 18 - 33%

Sodium citrate dihydrate 18 - 33%

Sodium stearate 0 - 2.5%

Sodium perborate monohydrate 0 - 13%

Tetraacetyl ethylene diamine (TAED) 0 - 8%

Maleic acid/acrylic acid copolymer 4 - 8%

Enzymes 0.0001 - 0.1%

Liquid automatic dishwashing composition containing protected bleach particles XI

Sodium silicate - 10% 64

XII: Automatic dishwashing compositions as described in I , II, III, IV, VI and X, wherein perborate is replaced by per- carbonate .

XIII: Automatic dishwashing compositions as described in I-VI, which additionally contain a manganese catalyst. The manganese catalyst may, e.g., be one of the compounds described in "Efficient manganese catalysts for low-temperature bleaching", Nature, (1994), 369, 637-639.

MATERIALS AND METHODS

TEXTILES

WFKION standard textile pieces (egg stains) were obtained from WFK Testgewebe GmbH, Christenfeld 10, D-41379 Brϋggen-Bracht , Germany .

STRAINS :

B . subtilis DN1885 (Diderichsen et al . , 1990) 65

B . lentus 309 and 147 are specific strains of Bacillus lentus, deposited with the NCIB and accorded the accession numbers NCIB 10309 and 10147, and described in US Patent No. 3,723,250 incorporated by reference herein.

E. coli MC 1000 (M.J. Casadaban and S.N. Cohen (1980); J. Moi .

Biol . 138 179-207), was made r^~,m⁺ by conventional methods and is also described in US Patent Application Serial No. 039,298.

PLASMIDS: pJS3 (SEQ ID NO:60): E. coli - B . subtilis shuttle vector containing a synthetic gene encoding for subtilase 309 (Described by Jacob Schiødt et al . in Protein and Peptide letters 3:39-44 (1996)).

pSX222 : B . subtilis expression vector (described in WO 96/34946) .

GENERAL MOLECULAR BIOLOGY METHODS: Unless otherwise mentioned the DNA manipulations and transformations were performed using standard methods of molecular biology (Sambrook et al . (1989) Molecular cloning: A laboratory manual, Cold Spring Harbor lab., Cold Spring Harbor, NY; Ausubel, F. M. et al . (eds.) "Current protocols in Molecular Biology". John Wiley and Sons, 1995; Harwood, C. R. , and Cutting, S. M. (eds.) "Molecular Biological Methods for Bacillus". John Wiley and Sons, 1990) .

Enzymes for DNA manipulations were used according to the specifications of the suppliers.

ENZYMES FOR DNA MANIPULATIONS 66

Unless otherwise mentioned all enzymes for DNA manipulations, such as e.g. restiction endonucleases, ligases etc., are obtained from New England Biolabs, Inc.

PROTEOLYTIC ACTIVITY

In the context of this invention proteolytic activity is expressed in Kilo NOVO Protease Units (KNPU) . The activity is

® determined relatively to an enzyme standard (SAVINASE ) , and the determination is based on the digestion of a dimethyl casein (DMC) solution by the proteolytic enzyme at standard conditions, i.e. 50°C, pH 8.3 , 9 min. reaction time, 3 min. measuring time. A folder AF 220/1 is available upon request to Novo Nordisk A/S, Denmark, which folder is hereby included by reference.

A GU is a Glycine Unit, defined as the proteolytic enzyme activity which, under standard conditions, during a 15 minutes' incubation at 40°C, with N-acetyl casein as substrate, produces an amount of NH₂-group equivalent to 1 mmole of glycine.

Enzyme activity can also be measured using the PNA assay, according to reaction with the soluble substrate succinyl- alanine-alanine-proline-phenyl-alanine-para-nitro-phenol , which is described in the Journal of American Oil Chemists Society, Rothgeb, T.M., Goodlander, B.D., Garrison, P.H., and Smith, L.A. , (1988) .

FERMENTATION:

Fermentations for the production of subtilase enzymes were performed at 30°C on a rotary shaking table (300 r.p.m.) in 500 ml baffled Erlenmeyer flasks containing 100 ml BPX medium for 5 days . 67

Consequently in order to make an e.g. 2 liter broth 20 Erlenmeyer flasks were fermented simultaneously.

MEDIA: BPX Medium Composition (per liter)

Potato starch 100 g

Ground barley 50 g

Soybean flour 20 g

Na₂HP0₄ x 12 H₂0 9 g

Pluronic 0.1 g

Sodium caseinate 10 g

The starch in the medium is liquefied with α-amylase and the medium is sterilized by heating at 120°C for 45 minutes. After sterilization the pH of the medium is adjusted to 9 by addition of NaHC0₃ to 0.1 M.

EXAMPLE 1

CONSTRUCTION AND EXPRESSION OF ENZYME VARIANTS:

SITE-DIRECTED MUTAGENESIS:

Subtilase 309 (savinase^®) site-directed variants of the invention comprising specific insertions and comprising specific substitutions were made by traditional cloning of DNA fragments (Sambrook et al . , Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor, 1989) produced by PCR with oligos containing the desired insertions (see below) .

The template plasmid DNA was pJS3 (see below) , or an analogue of this containing a variant of Subtilase 309. 68

Insertions and substitutions were introduced by oligo directed mutagenesis to the construction of variants.

The Subtilase 309 variants were transformed into E. coli . DNA purified from a over night culture of these transformants were transformed into B . subtilis by restriction endonuclease digestion, purification of DNA fragments, ligation, transformation of B. subtilis . Transformation of B. subtilis was performed as described by Dubnau et al . , 1971, J. Moi. Biol. 56, pp. 209- 221.

SITE-DIRECTED MUTAGENESIS IN ORDER TO INTRODUCE INSERTIONS AND SUBSTITUTIONS IN A SPECIFIC REGION:

The overall strategy to used to perform site-directed mutagenesis was:

Mutagenic primers (oligonucleotides) were synthesized corresponding to the DNA sequence flanking the sites of insertion and substitutions, separated by the DNA base pairs defining the insertions and substitutions.

Subsequently, the resulting mutagenic primers were used in a PCR reaction with the modified plasmid pJS3 (see above) . The resulting PCR fragment was purified and extended in a second PCR-reaction, the resulting PCR product was purified and either cloned into the E. coli - B . subtilis shuttle vector (see below) or extended in a third PCR-reaction before being digested by endonucleases and cloned into the E. coli - B . subtilis shuttle vector (see below) . The PCR reactions are performed under normal conditions .

Following this strategy insertions and substitutions was constructed in savinase^® wherein insertions and substitutions 69 was introduced according to the below table. The primers used for each PCR step are shown as well as the cloning sites used.

Following the above strategy a detailed example follows:

Two insertion and one substitution was constructed in savinase^® wherein insertions was introduced in position 99 (*99aD) and 217 (*217aP) respectively and a substitution was introduced in position S99A (see below) .

The insertion and substitution at position 99 was introduced by a mutagenic primer (5' CCG AAC CTG AAC CAT CCG CGG CCC CTA GGA CTT TAA CAG C 3' ( sense) ) (SEQ ID NO: 71) were used in a PCR reaction with an opposite primer (5' GAG TTA AGC CCA GAA GAT GTG GAC GCG 3' (antisense)) (SEQ ID NO:83) .

The produced PCR fragment were extended towards the C-terminal of Savinase by a second round of PCR introducing the insertion at position 217 with primer; 5' CAT CGA TGT ACC GTT TGG TAA GCT GGC ATA TGT TG 3' (SEQ ID NO: 94) . The second round PCR product were extended towards the C-terminal of Savinase by a third round of PCR with primer; 5' AAC CGC ACA GCG TTT TTT TAT TGA TTA ACG CGT TGC 3' (SEQ ID NO.105), situated downstream at the Mlu I site in pJS3. All PCR reactions used plasmid pJS3 as template. The extended DNA- fragment resulting from third round PCR was cloned into the Sal I- and Mlu I- sites of the modified plasmid pJS3 (see above) .

The plasmid DNA was transformed into E. coli by well-known techniques and one E. coli colony were sequenced to confirm the mutation designed.

All other variants were constructed in an analogous manner.

In order to purify a subtilase variant of the invention, the B . subtilis pJS3 expression plasmid comprising a variant of the 70 invention was transformed into a competent B . subtilis strain and was fermented as described above in a medium containing 10 μg/ml Chloramphenicol (CAM) .

Primers and cloning sites:

71

72

73

74

75

76

77

EXAMPLE 2

PURIFICATION OF ENZYME VARIANTS:

This procedure relates to purification of a 2 liter scale fermentation for the production of the subtilases of the invention in a Bacillus host cell.

Approximately 1.6 liters of fermentation broth were centrifuged at 5000 rpm for 35 minutes in 1 liter beakers. The supernatants were adjusted to pH 6.5 using 10% acetic acid and filtered on Seitz Supra S100 filter plates.

The filtrates were concentrated to approximately 400 ml using an A icon CH2A UF unit equipped with an Amicon S1Y10 UF cartridge. The UF concentrate was centrifuged and filtered prior to absorption at room temperature on a Bacitracin affinity column at pH 7. The protease was eluted from the Bacitracin column at 78 room temperature using 25% 2-propanol and 1 M sodium chloride in a buffer solution with 0.01 dimethylglutaric acid, 0.1 M boric acid and 0.002 M calcium chloride adjusted to pH 7.

The fractions with protease activity from the Bacitracin purification step were combined and applied to a 750 ml Sephadex G25 column (5 cm dia.) equilibrated with a buffer containing 0.01 dimethylglutaric acid, 0.2 M boric acid and 0.002 m calcium chloride adjusted to pH 6.5.

Fractions with proteolytic activity from the Sephadex G25 column were combined and applied to a 150 ml CM Sepharose CL 6B cation exchange column (5 cm dia.) equilibrated with a buffer containing 0.01 M dimethylglutaric acid, 0.2 M boric acid, and 0.002 M calcium chloride adjusted to pH 6.5.

The protease was eluted using a linear gradient of 0-0.1 M sodium chloride in 2 litres of the same buffer (0-0.2 M sodium chloride in case of Subtilisin 147) .

In a final purification step protease containing fractions from the CM Sepharose column were combined and concentrated in an Amicon ultrafiltration cell equipped with a GR81PP membrane (from the Danish Sugar Factories Inc.) .

By using the techniques of Example 1 for the construction and fermentation, and the above isolation procedure the following subtilisin 309 variants were produced and isolated: 79

Position 96 insertion variants:

L96LA

L96LA + A98T + A108C + A138C

Position 97 insertion variants:

G97GI + S99T

Position 98 insertion variants:

A98AS + A133E + T143K

A98AT + G97D A98ATGTG

A98AG

A98AS + R45K + S105G

A98AT + G97E

A98ASGTG A98AP + A98G + S99A

A98AT + Y167A + R170S + A194P

A98AI+A98G+S99H+G100S+S101A

Position 99 insertion variants:

S99SD + S99A

S99SA

S99SE + S99T

S99SD + S99A + A133E S99SD + S99A + T143K

S99SD

S99SE

S99SD 4- S99A + S216SP

S99SD + S99A + S216SDP S99SD + S99A + P129PD

S99SD + S99A + P129PR

S99SD + S99A + L217F + A228V + A230V S99SD + S99A + L217LP S99SD + S99A + D42DN S99SR + S99T S99SQ + S99T S99SD + M222S

S99SD + N76D + A194P + A230V

S99SN

S99SD + S99A + P131T

EXAMPLE 3

The "MODEL DETERGENT WASH PERFORMANCE TEST" :

In order to asses the wash performance of selected subtilase variants in a standard detergent composition, standard washing experiments may be performed using the below experimental conditions:

Detergent : Model detergent

Detergent dosage 4.0 g/1 pH 10.1 Wash time 20 min

Temperature : 30°C

Water hardness: 15°dH

Enzyme concentration: 10 nm (in the detergent solution)

Test system: 10 ml beakers with a stirring rod Textile/volume: 5 textile pieces (0 2.5 cm) /50 ml detergent solution

Test material : WFKION (egg stains)

The composition of the model detergent is as follows

6.2% LAS (Nansa 80S)

2 % Sodium salt of C₁₆- C₁₈ fatty acid 81

4% Non-ionic surfactant (Plurafax LF404)

22% Zeolite P

10.5% Na₂C0₃

4% Na₂Si₂0₅ 2% Carboxymethylcellulose (CMC)

6.8% Acrylate liquid CP5 40%

20% Sodium perborate (empirical formula NaB0₂.H₂0₂)

0.2% EDTA

21% Na₂S0₄ Water (balance)

pH of the detergent solution is adjusted to 10.1 by addition of HCl or NaOH. Water hardness is adjusted to 15°dH by addition of CaCl₂ and MGC1₂ (Ca²⁺:Mg²⁺ = 4:1) to the test system. After washing the textile pieces were flushed in tap water and air- dried.

Measurement of the reflectance (R_varιant) on the test material is performed at 460 nm using a Macbeth ColorEye 7000 photometer (Macbeth, Division of Kollmorgen Instruments Corporation,

Germany) . The measurements are performed accordance with the manufacturer's protocol.

In order to determine a blank value, a similar wash experiment is performed without addition of enzyme. The subsequent measurement of the reflectance (R_bιank) is performed as described right above .

A reference experiment is then performed as described above, wherein the wash performance of the parent enzyme is tested. The subsequent measurement of the reflectance (R_parent*¹ -i*³ performed as described right above. 82

The wash performance is evaluated by means of the Performance Factor (P) which is defined in accordance with the below formula :

^■P ^_ ■ ^■'"^•■variant -R-blan *' * -^parent R-blank *'

= R„ R, parent

Using the above test method the following results were obtained:

As it appears, the subtilase variants exhibit improved wash performance on egg stains in comparison to the parent subtilase, i.e. Savinase .

EXAMPLE 4

THE "OVO-INHIBITION ASSAY" 83

The below inhibition assay is based on the principle that the subtilase variant to be tested will catalyse the hydrolysis of a peptide-pNA bond, thereby releasing the yellow pNA, which may conveniently be followed at 405 nm. The amount of released pNA after a given period of time is a direct measure of the subtilase activity. By carrying out such hydrolysis experiments with and without inhibitor, respectively, it is possible to obtain a quantitative measure for the degree to which a certain subtilase variant is inhibited.

Reaction conditions :

Enzyme concentration: 0.0003 mg/ml

Cone, of trypsin inhibitor type IV- 0 0.0015 mg/ml

Initial substrate concentration: 0.81 mM

Reaction time: 11 min

Assay temperature : 25°C

Assay pH: 8.6

Absorbance measured at : 405 nm

Assay solutions:

Substrate solution (2 mM) : 500 mg Suc-Ala-Ala-Pro-Phe-pNA is dissolved in 4 ml DMSO (200 mM) . This solution is diluted 100 times with the buffer solution described below. The concentration of substrate in the resulting substrate solution is 2 mM.

Inhibitor solution (0.005 mg/ml): 5 mg trypsin inhibitor type IV-0 (Sigma T-1886) is dissolved in 10 ml water. This solution is dissolved 100 times with the buffer solution described below. The concentration of inhibitor in the resulting inhibitor solu- tion is 0.005 mg/ml. 84

Enzyme solution (0.001 mg/ml) : 1 mg enzyme is dissolved in 10 ml water. This solution is dissolved 100 times with the buffer solution described below. The concentration of enzyme in the resulting enzyme solution is 0.001 mg/ml.

Buffer solution (pH 8.6) : 15.7 mg Tris is dissolved in an appropriate amount of water and 0.75 ml 30% (w/v) BRIJ (BRIJ 35 poly- oxyethylenelaurylether, 30% (w/v), Sigma Cat. No. 430AG-6) is added. The pH is adjusted to 8.6 with 4 M NaOH and the solution is diluted to 1 liter with water.

Assay with inhibitor

1 volume unit (e.g. 80 μl) inhibitor solution is mixed with 1 volume unit (e.g. 80 μl) enzyme solution in an appropriate reac- tion vessel (e.g. a spectrophotometer cell or a micro titer plate) and equilibrated at 25°C for 15 min. 1.375 volume units (e.g. 110 μl) substrate solution is added to the reaction vessel after which the absorbance at 405 nm is followed for 11 min (e.g. by measuring every 10^th or 30^th second) . The slope of the absorbance curve is calculated using linear regression analysis. The slope of the absorbance curve is denoted α_inhibltor.

Assay without inhibitor

1 volume unit (e.g. 80 μl) buffer solution is mixed with 1 vol- ume unit (e.g. 80 μl) enzyme solution in an appropriate reaction vessel (e.g. a spectrophotometer cell or a micro titer plate) and equilibrated at 25°C for 15 min. 1.375 volume units (e.g. 110 μl) substrate solution is added to the reaction vessel after which the absorbance at 405 nm is followed for 11 min (e.g. by measuring every 10^th or 30^th second) . The slope of the absorbance 85 curve is calculated using linear regression analysis. The slope of the absorbance curve is denoted α.

Blank

1 volume unit (e.g. 80 μl) inhibitor solution is mixed with 1 volume unit (e.g. 80 μl) buffer solution in an appropriate reaction vessel (e.g. a spectrophotometer cell or a micro titer plate) and equilibrated at 25°C for 15 min. 1.375 volume units (e.g. 110 μl) substrate solution is added to the reaction vessel after which the absorbance at 405 nm is followed for 11 min. These measurements are not used in the calculations, but merely serve as a control that no enzyme has been added to the buffer and/or substrate solution.

Calculation of Residual Activity (RA)

The residual enzyme activity (RA) is calculated according to the below formula:

RA = (c nhibitor /α) x 100=

Using the above test, the following results were obtained:

8 6

As it appears, the subtilase variants were inhibited to a much smaller extent than the parent subtilase, i.e. savinase^*.

EXAMPLE 5

Performance of the subtilase variant of the invention in Automatic Dishwashing (ADW)

The performance of the variant of the invention in ADW was tested in a commercial available household dishwash composition (Somat Turbo, from Henkel Washmittel GmbH) using standard conditions. The soil used was an egg/milk mixture coated on a steel plate. Further, a ballast soil containing various foodstuffs was added.

Detergent : Somat Turbo

Detergent dosage 4.0 g/1 pH 10.7 (as is)

Water hardness : 3°dH (machine ion exchanger) 17

Temperature : 55°C

Enzyme concentration: 20 nM and 40 nM, based on the total volume of wash water in the machine

Test method: Egg/milk soiling on steel plates as described below

Machine : Cylinda Compact Wash program: Program 4 without pre- flush

Materials 220 ml full cream milk 15 eggs, medium size Steel plates, diameter 18 cm

The Somat Turbo dishwash composition was heated at 85°C for 5 minutes in a microwave oven in order to inactivate enzyme activity in the composition.

Soiling of steel plates

220 ml full cream milk was mixed with 15 raw eggs in a Braun UK 20 kitchen machine for 2 minutes, After sieving, stainless steel plates were soiled in the mixture by immersion.

The plates were dried overnight at room temperature in an upright position. The dried plates were then heated at 120°C for 45 minutes in order to denature the proteins on the surface.

ADW experiments

For each experiment, 10 soiled plates were washed without pre- wash (Program 4) in a Cylinda Compact machine. In addition to the soiled plates, the machine was filled up with 10 porcelain plates, 4 glasses, 4 cups and 16 pieces of cutlery. Furthermore, 50 g of ballast slurry was added to the machine. The composition of the slurry was as follows:

Potato starch (5.43%), wheat flour (4.38%), vegetable oil (4.32%), margarine (4.32%), lard (4.32%), cream (8.76%), full cream milk (8.76%), eggs (8.76%), tomato ketchup (3.00%), barbecue sauce (2.19%), mustard (4.00%), benzoic acid (0.73%), water (3 mM Ca²⁺ + Mg²⁺) (36.71%).

Measurements and calculations

The light reflection values (R-values) were measured at six different locations on the plates using a Minolta Chroma Meter (Type: CR-300) . Measurements were made on clean plates (R_clean) , on soiled plates after heating (R_solled) and on plates after wash

⁽R.after was_h)

The removed protein film (%RPF) was calculated according to the below formula:

%RPF - 100 % X ( R_{after wash} - R_solie_d ) ( Rclean ~^{~ R}soιled )

Using the above test method the following results were obtained (± indicates the standard deviation) :

As it appears, the variant of the invention has a superior performance as compared to Savinase^® . 8 9

EXAMPLE 6

Wash performance of the subtilase variant of the invention in a commercially available powder detergent

In order to assess the wash performance of selected subtilase variants in a commercial detergent composition, standard washing experiments were performed using the below experimental conditions :

90

Detergent dosage : 4 g/ 1

Wash temperature : 30°C

Washing time: 20 minutes

Water hardness : 15°dH (Ca²⁺ : Mg²⁺ = 4 : 1 ) pH: Not adjusted

Enzyme concentrations 1, 2, 5, 10, 30 nM

Test system: 150 ml glass beakers with a stirring rod

Textile/volume : 5 textile pieces (0 2.5 cm) in 50 ml detergent

Test material : WFKION (egg stains)

The detergent used was obtained from supermarket in Germany (Persil Megapearls) . Prior to use all enzymatic activity was in the detergents were inactivated by microwave treatment (5 minutes, 85°C) .

The reflectance measurements were performed as described in Example 3 herein.

The data (the R values) were evaluated as follows:

A variant having a higher R-value than savinase^® was given the value 1.

A variant having a lower R-value than savinase^® was given the value -1.

A variant having a R-value similar to savinase^® was given the value 0. 91 Results :

Variant Value

Savinase^® 0 L96LA 1

L96LA+A98T+A108C+A138C 1

G97GI+S99T 1

As I appears, the subtilase variants exhibit improved wash performance in a commercial detergent as compared to savinase^®

92 CLAIMS

1. Use of a subtilase variant for removal of egg stains from laundry or from hard surfaces, the subtilase variant coπprising at least one addi- tional amino acid residue in the active site loop (b) region from position 95 to 103 (BASBPN numbering) .

2 . Use according to claim 1 , wherein the additional amino acid residue has been inserted in a position selected from the group consisting of : between positions 95 and 96, between positions 96 and 97, between positions 97 and 98, between positions 98 and 99, between positions 99 and 100 , between positions 100 and 101, between positions 101 and 102 , between positions 102 and 103 , between position 103 and 104 and combinations thereof .

3. Use according to claim 2, wherein the additional amino acid residue has been inserted in a position selected from the group consisting of : between positions 98 and 99 and between positions 99 and 100.

4 . Use according to any of claims 1 - 3 , where the variant - when tested in the "Ovo- inhibition Assay" disclosed in Example 4 herein - has a Residual Activity of at least 10% , such as at least 15% , preferably at least 20% , more preferably at least 25% .

5. Use according to any of claims 1-4 , wherein the insertion between positions 98 and 99 is selected from the group consisting of X98XA, X98XT, X98XG and X98XS .

6 . Use according to any of claims 1 -4 , wherein the insertion between positions 99 and 100 is selected from the group consisting of X99XD , X99XE , X99XK and X99XR . 93

7. Use according to any of the preceding claims, wherein the variant comprises at least one further modification.

8. Use according to claim 7, wherein the further modification is performed in a position selected from the group consisting of: substitution in position 99, substitution in position 133, substitution in position 143, substitution in position 167, substitution in position 170, substitution in position 194, insertion between positions 42 and 43, insertion between positions 129 and 130, insertion between positions 216 and 217, insertion between 217 and 218, and combinations thereof.

9. Use according to claim 8, wherein the variant is selected from the group consisting of

a variant coπprising an insertion of at least one additional amino acid residue between positions 98 and 99 and further coπprising a substitution in positions 133 and 143,

a variant coπprising an insertion of at least one additional amino acid residue between positions 99 and 100 and further coπprising a substitution in position 99,

a variant coπprising an insertion of at least one additional amino acid residue between positions 98 and 99 and further coπprising substitutions in positions 167, 170 and 194,

a variant coπprising an insertion of at least one additional amino acid residue between positions 99 and 100 and further coπprising an insertion of at least one additional amino acid residue between positions 216 and 217, 94 a variant coπprising an insertion of at least one additional amino acid residue between positions 99 and 100 and further coπprising an insertion of at least one additional amino acid residue between positions 217 and 218,

a variant coπprising an insertion of at least one additional amino acid residue between positions 99 and 100 and further coπprising an insertion of at least one additional amino acid residue between positions 42 and 43, and

a variant coπprising an insertion of at least one additional amino acid residue between positions 99 and 100 and further coπprising an insertion of at least one additional amino acid residue between positions 129 and 130.

10. Use according to any of the preceding claims, wherein the parent subtilase belongs to the sub-group I-Sl.

11. Use according to claim 10, wherein the parent subtilase is selected from the group consisting of BSS168, BASBPN, BSSDY, and BLSCAR, or functional variants thereof having retained the characteristic of sub-group I-Sl.

12. Use according to any of claims 1-9, wherein the parent subtilase belongs to the sub-group I-S2.

13. Use according to claim 12, wherein the parent subtilase is selected from the group consisting of BLS147, BLSAVI, BAPB92, TVTHER and BYSYAB, or functional variants thereof having retained the characteristic of sub-group I-S2. 95

14. Use according to claim 13, wherein the parent subtilase is BLSAVI (SEQ ID NO: 1) .

15. Use according to claim 14, wherein the variant is S99SD + S99A.

16. Use according to claim 14, wherein the variant is S99SR + S99T.

17. Use according to claim 14, wherein the variant is A98AS + A133E + T143K.

18. Use according to claim 14, wherein the variant is A98AT + Y167A 4- R170S + A194P.

19. Use according to claim 14, wherein the variant is S99SD + S99A + P129PD.

20. Use according to claim 14, wherein the variant is S99SD + S99A + S216SP.

21. Use according to claim 14, wherein the variant is S99SD + S99A + S216SDP.

22. Use according to claim 14, wherein the variant is S99SD + S99SA + L217LP.

23. Use according to claim 14, wherein the variant is S99SD + D42DN.

24. Use according to claim 14, wherein the variant is S99SD + S99A + D42DN. 96

25. A subtilase variant selected from the group consisting of

a variant comprising at least one additional amino acid residue in the ac- tive site (b) loop corresponding to the insertion of at least one additional amino acid residue between positions 98 and 99 and further coπprising at least one additional modification (BASBPN numbering) , and

a variant comprising at least one additional amino acid residue in the ac- tive site (b) loop corresponding to the insertion of at least one additional amino acid residue between positions 99 and 100 and further comprising at least one additional modification (BASBPN numbering) ,

where the variant - when tested in the "Ovo-inhibition Assay" dis- closed in Example 4 herein - has a Residual Activity of at least 10%.

26. The variant according to claim 25, where the variant has a Residual Activity of at least 15%, preferably at least 20%, more preferably at least 25%.

27. The variant according to claims 25 and 26 having the characteristic as defined in any of claims 9-24

28. A subtilase variant as defined in any of claims 9-24.

29. An isolated DNA sequence encoding a subtilase variant as defined in any of claims 25-28.

30. An expression vector coπprising the isolated DNA sequence of claim 29. 97

31. A microbial host cell transformed with the expression vector of claim 30.

32. A microbial host cell according to claim 31, which is a bacterium, preferably a Bacillus, especially a B. lentus.

33. A microbial host cell according to claim 31, which is a fungus or yeast, preferably a filamentous fungus, especially an Aspergillus .

34. A method for producing a subtilase variant according to any of claims 25-27, wherein a host according to any claims 31-33 is cultured under conditions conducive to the expression and secretion of said variant, and the variant is recovered.

35 . A cleaning or detergent composition, preferably a laundry or dishwash composition, comprising the variant according to any of claims 25 -28 .

36 . A composition according to claim 35 , which additionally comprises a cellulase , a lipase , a cutinase , an oxidoreductase , another protease , an amylase or a mixture thereof .

37 . Use of a variant as defined in any of claims 25 -28 in a cleaning or detergent composition, preferably a laundry and/or a dishwash composition .

38 . A method for removal of egg stains from a hard surface or from laundry, the method comprising contacting the egg stain- containing hard surface or the egg stain-containing laundry with a cleaning or detergent composition , preferably a laundry or dishwash composition, containing a subtilase variant comprising 98 at least one additional amino acid residue in the active site loop (b) region from position 95 to 103 (BASBPN numbering) .

39. A method according to claim 38, wherein the variant has the characteristics as defined in any of claims 2-24.

40. A method according to any of claims 38-39, wherein the composition additionally comprises a cellulase, a lipase, a cutinase, an oxidoreductase, another protease, an amylase or a mixture thereof .

41. Use of a cleaning or detergent composition, preferably a laundry or dishwash composition, containing a subtilase variant comprising at least one additional amino acid residue in the active site loop (b) region from position 95 to 103 (BASBPN numbering) for removal of egg stains from laundry or from hard surfaces.

42. Use according to claim 41, wherein the variant has the characteristics as defined in any of claims 2-24.

43. Use according to any of claims 41-42, wherein the composition additionally comprises a cellulase, a lipase, a cutinase, an oxidoreductase, another protease, an amylase or a mixture thereof .